橋本 直子

コルチゾール産生腫瘍では、PRKACA、Wnt、GNAS変異などが知られているが、分子生物学的全容は解明されていない。今回、クッシング症候群(CS)とサブクリニカルクッシング症候群(SCS)の特性を知るために、原発性アルドステロン症(APA)症例を比較対照としてシングルセル解析を行った。対象は、当院で2020～2021年に副腎腫瘍摘出術を施行され病理診断されたCS 2例、SCS 5例、APA 5例とした。全ての摘出副腎組織から得られた細胞をクラスター分析した結果、23のクラスターに分類することができた。各クラスターがそれぞれどのような細胞集団であるのか、細胞マーカを用いて同定し、それらを分離することで、副腎皮質細胞のみを抽出することができた。腫瘍部における各細胞集団の割合をみると、腫瘍部であっても腫瘍コンテンツは約35%にとどまることが分かった。副腎皮質細胞と同定された細胞群をサブクラスタリング解析すると、SCS腫瘍細胞が多くを占めるサブクラスターや、APA細胞が多くを占めるサブクラスターが存在し、サブクラスターごとにCYP11B1やCYP17A1の発現パターンが異なっていた。

当院でオシロドロスタットを投与した副腎性クッシング症候群3例の治療成績を報告した。原疾患の内訳は副腎腺腫が2例、PMAHが1例であった。投与期間は3ヵ月～1年6ヵ月で、3例とも尿中コルチゾール排泄量が速やかに減少し、正常化した。副腎腺腫の2例に対してはオシロドロスタット投与後に副腎摘除術を行い、完治した。

シングルセル解析を用いて、分子基盤や細胞間相互作用など、腫瘍内微小環境の探索を行った。非機能性下垂体腺腫(NF)8例、成長ホルモン産生下垂体腺腫(GHoma)3例、正常下垂体前葉組織(normal)4例を対象とした。手術で摘出された組織をcollagenaseを用いてdigestionした後、死細胞除去を行い、Chromium Controllerでemulsion作成/逆転写を行いシークエンスした。計45942細胞に対して解析を行った。クラスター細胞種同定と構成細胞特性の解析では、45942細胞は八つのmajorクラスターに大別され、組織を構成する細胞の割合をみてみると、マクロファージなどの貪食細胞を中心とする免疫細胞が6割ほどを占めていた。Endocrine componentのみを抽出して下流解析を行った。その結果、各クラスターはNF/GHoma/normalのいずれかの細胞が大多数を占めていた。cell-cell interaction解析の結果、GHとnormalでは類似したligand発現を認め、NFでは腫瘍増殖にかかわるMDK signaling pathwayが特異的にみられた。NF/GHoma/normalの腫瘍細胞または正常前葉細胞が受けるシグナルに着目すると、正常前葉細胞にみられたGH、PRL受容体の発現がNFとGHomaの腫瘍細胞には認められなかった。

過去12年5ヵ月間の膵神経内分泌腫瘍(NEN)切除例35例(手術時年齢56.7±16.3歳)の標本を用い、免疫染色scoring、遺伝子解析によりNENの分子生物学的特性を検討した。免疫染色はKi-67、クロモグラニンA、インスリン、ガストリン、グルカゴン、ソマトスタチン、SSTR2、SSTR3、SSTR5、DAXX、ATRX、PAX6に行った。その結果、主成分分析より、機能性ガストリノーマは悪性度の高いNECやMiNENと類似した性質を持つことが示された。また、腫瘍増殖能が高いほどSSTR2とSSTR5は低発現で、腫瘍最大径が大きいほどSSTR5が低発現でKi-67は高発現であった。再発・転移、原病死症例は無再発生存症例に比してKi-67が有意に高値でDAXX、ATRXに有意差はなかった。以上より、Ki-67、ソフトスタチンレセプターやDAXX、ATRXの発現レベルの違いは、予後や転移の予測因子となり得ることが示唆された。

<title>Abstract</title> In many cancers, including hormone sensitive tumors such as breast cancer, the “gain of function” caused by mutations in the tumor suppressor gene p53 plays an important role in the acquisition of malignant phenotypes and the regulation of cancer stem cell characteristics. However, its action of molecular mechanisms, particularly in vivo conditions, has not been fully clarified. Therefore, we focused on the “gain of function” of mutant p53 and the cholesterol biosynthesis pathway, especially the mevalonate(MVA) pathway, using breast cancer cells, and clarified the interaction between them and the relationship with cancer malignancy using 3D-culture. Here, we generated knock out and knock in breast cancer cell lines for p53 using CRISPR-Cas9 system, and then confirmed malignant morphological changes by 3D-culture model. We found that the introduction of mutant p53 was solely able to mediate the malignant transformation of cancer. Next, focusing on the relationship between cancer malignant transformation and lipid metabolism pathway, we investigated the role of the MVA pathway in malignant transformation by mutation p53. When investigating the effects of the addition of HMG-CoA inhibitors and isoprenoids, intermediate metabolites were important for malignant transformation during 3D culture. Furthermore, knockdown of SREBP2, which controls the MVA pathway, suppressed malignant phenotypes, so we proceeded with analysis of the interaction between mutant p53 and SREBP2. As the result, we found that mutant p53 and SREBP2 co-localize in the nucleus and consistently mutant p53 was associated with mevalonate pathway genes in parallel with binding pattern of SREBP2. Thus, our results provide the novel insight into the potential therapeutic targets for breast cancer with poor prognosis.

<title>Abstract</title> p53 is mutated more than half of human cancers, and mutant p53, a gain of function, can actively have functional consequences with tumorigenesis. However, its action of molecular mechanisms, particularly in vivo conditions, has not been fully are clarified. Here, we generated KO and KI (R280K) breast cancer cell lines for p53 using CRISPR/Cas9 system, and then performed a three-dimensional culture model. We found that the introduction of mutant p53 was solely able to mediate the transformation to poor architectural structure. Interestingly, our findings in statin-effect along with cholesterol synthesis pathway, especially isoprenoid dependency, revealed that this pathway is necessary and sufficient for the regulation of malignant architecture in SREBP2-dependent manner with cooperatively being controlled by mutant p53 on 3D-cultured breast cancer. Furthermore, in accordance with the malignancy progresses, SREBP2 was accumulated in nuclear and nuclear membrane portion with enhancement in malignant formation. In addition, we found that mutant p53 interacts with SREBP2, and consistently mutant p53 was associated with DHCR7 promoter in parallel with binding pattern of SREBP2. Thus, our results provide the novel insight into the mutant p53, a gain of function, and its linkage to poor architectural structure in 3D-cultured breast cancer cells via SREBP2-dependent isoprenoids regulation as potential therapeutic targets.

<title>Abstract</title> Iron is an essential cofactor for many proteins that function in electron transport or oxygen transport as heme or iron-sulfur cluster. On the contrary, iron also has the potential to cause oxidative damage if not carefully regulated and when in labial iron excess. Clinical studies show that elevated serum ferritin levels are observed in most patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this context, p53 is shown to induces some mitochondrial iron regulatory genes. The role of crosstalk between p53 and iron metabolism has not been sufficiently examined in the pathogenesis of diabetes and NAFLD. Here, we examined the role of ferredoxin reductase (FDXR), a key mitochondrial regulator for iron metabolism, as p53-inducible gene with focusing on the hepatocyte and liver. We confirmed that p53 induced FDXR expression in HepG2 cells and SKEHP1 cells. Biochemical analysis demonstrated that FDXR regulated ROS levels via iron metabolism. In vivo analysis, high-fat diet activated the p53-FDXR pathway in mice liver. We generated transgene expression in mice liver using adenovirus infection carrying shRNA or CRISPR Cas9 system. Treatment with the FDXR knockdown increased hepatic iron content and aggravated glucose intolerance. Besides, forkhead box protein O1 (FOXO1), a key transcriptional factor that induces phosphoenolpyruvate carboxylase and glucose-6-phosphatase increased ratio of nuclear localization, indicating hepatic gluconeogenesis activation. Consistently, biochemical analysis in HepG2 cells demonstrated that FDXR regulated insulin-dependent FOXO1 nuclear exclusion through oxidative stress. In conclusion, p53-inducible FDXR regulates iron metabolism and oxidative stress. FDXR inhibits iron accumulation and oxidative stress in liver and links to suppression of hepatic gluconeogenesis via insulin-dependent FOXO1 nuclear exclusion. The results of this study provide important new insights into relationship between iron metabolism and glucose metabolism as well as potentially identify novel therapeutic targets for the treatment of diabetes and NAFLD.

<title>Abstract</title> Objective: To clarify the relationship between proteomic expression and clinical feature of pituitary adenoma. Methods: We have previously developed non-target proteomics analysis, which enables to detect and quantify approximately 7,000 to 9,000 kinds of protein weave, in parallel with RNA-seq analysis, and then subjected to 14 cases of pituitary adenoma surgically removed at Chiba University Hospital. Bioinformatic evaluation including DEGs, heatmap and PCA analyses was performed to reveal underlying their molecular pathogenesis. Results: We successfully identified 789 differentially expressed proteins and 593 DEGs in non-target proteomics and RNA-seq, respectively. Intriguingly, PCA analysis demonstrated that tumors were clearly divided into 3 groups based on protein expression profile; functional pituitary adenomas consisting of two subtypes depending on Pit1 and T-pit linage, and non-functional tumors consisting of two distinct subtypes, with properties close to functional tumors and unique characteristics of hard tumor difficult to remove by endoscopic surgery. To address the underlying molecular biological functions in each group clustering analysis and heat-map were performed and we found that 3 groups were separated clearly with their own both gene and protein expression profile. Indeed, for instance, GO term of plasma membrane part was significantly enriched in hard tumor group, pathways of GH receptor signaling, GH hormone synthesis as in GH-positive group. Conclusions: We herein demonstrate that pituitary adenoma can be uniquely separated into certain categories through our novel non-target proteomics with coupling to RNA-seq, particularly providing novel group of hard tumor characteristics with enriched expression of both protein and mRNA in plasma membrane part. Thus our method would be beneficial and useful to elucidate underlying molecular pathogenesis for pituitary tumors, while further analysis is required.