中山 哲俊

褐色細胞腫6例(腫瘍検体5検体、正常副腎皮質5検体)を対象としてsingle cell RNA-seq解析を行った。全検体のすべての細胞のうち解析に足る細胞と考えられたのは102732個であった。これらは遺伝子発現の類似性から36のクラスターに分類され、腫瘍部と正常部とでは発現パターンが大きく異なっていた。症例ごとに細胞の発現パターンを観察すると、腫瘍組織に共通した傾向のみならず、症例ごとに異なる遺伝子発現をもつことも推測された。髄質細胞と同定されたもののみに限定して遺伝子発現を観察したところ、腫瘍細胞は7つのクラスター(0～6)に分類された。このうちクラスター0は細胞数が最も多く、CHGAなど褐色細胞腫で良く知られた分子が強く発現していた。クラスター0の特性を調べるため、monocle3を用いた遺伝子module解析を行ったところ、Module6の遺伝子群が本クラスターと強く相関することが分かった。他のクラスターの特性を同様の方法で調べたところ、クラスター1はExtracellular matrixの産生を担っていることが明らかになった。

<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.