Introduction

Pancreatic cancer is the fourth leading cause of cancer death in the western world and shows the worst mortality among common malignancies with a 5-year survival rate of lower than 5%. To identify novel therapeutic targets for aggressive and therapy-resistant pancreatic cancer, we here focus on the characterization of a novel gene C12orf48 (Chromosome 12 open reading frame 48) which was found to be trans-activated in pancreatic ductal adenocarcinoma (PDAC) cells according to our previous microarray expression profile. We demonstrate that C12orf48 protein can interact with PARP-1 directly and be involved in the repair of DNA breaks through enhancing PARP-1 activity. Thus, we termed this molecule PARP-1 binding protein (PARPBP).

Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme, catalyzes the transfer of the ADP-ribose unit from its substrate, NAD+, to some protein acceptors such as histones, p53, and PARP-1 itself. PARP-1, a molecular nick-sensor of DNA breaks, is essential in the repair of both DNA single-strand breaks (SSB) as well as double-strand breaks (DSB). PARP-1 is involved in multiple cellular processes including DNA repair, transcriptional regulation, chromatin modification, cell cycle progression, or genomic stability. Inhibition of PARP-1 enhanced the cytotoxicity of DNA-damaging agents and seemed to overcome one of the causes of resistance in cancer cells to anticancer treatment.

Materials and Methods

Cell Lines In our studies, we mainly used KLM-1 and SUIT-2 PDAC cell lines, which highly expressed C12orf48 proteins.

Immunoprecipitation and Mass-Spectrometric Analysis Protein bands that specifically observed in the cell extracts transfected with pCAGGS Flag-C12orf48-HA were excised and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS).

In-vitro PARP-1 Auto-Poly(ADP-ribosyl)ation Assays Briefly, purified C12orf48 recombinant protein and recombinant human PARP-1 were incubated in binding buffer (10mM Tris-HCl, pH 7.5, 1mM MgCl2, 1mM DTT) plus sonicated DNA. The reactions were started by adding 32P-labeled NAD+, and terminated with SDS sample buffer. Incorporation of 32P-labeled NAD+ to poly(ADP-ribosyl)ated proteins was visualized by autoradiography.

PARP-1 Activity in Cell Extracts PARP-1 activities in cell extracts were assayed using the universal colorimetric PARP assay kit based on the incorporation of biotinylated ADP-ribose onto histone H1 proteins. Briefly, cell extracts were loaded into a 96-well plate coated with histone H1, and incubated with biotinylated poly(ADP-ribose) and nicked DNA, size of which are 200-500 base pairs that are considered to be optimal for the PARP activation for 1 hour. Streptavidin-HRP (horseradish peroxidase) and TACS-SapphireTM was added subsequently to develop colors, and then the reaction was stopped by addition of 5% phosphoric acid. Finally, the absorbance was measured at 450nm in a spectrometrophotometer.

Sensitivity to DNA Damage Shcontrol and shC12orf48 KLM-1 cells were seeded into 6-well plates (5×105 cells/well), and incubated with indicated concentrations of adriamycin for 24 hours, H2O2 for 6 hours, or exposed to indicated intensity of UV radiation, and then incubated for 24 hours. Cell viability was measured using Cell-counting kit-8, and then the absorbance was measured at 490nm, and at 630nm with a Microplate Reader 550.

Results and Discussion

C12orf48, the gene trans-activated in pancreatic ductal adenocarcinoma (PDAC) cells through our genome-wide microarray analysis was confirmed to be overexpressed in five of the nine pancreatic cancer cases examined. Northern blot analysis using the C12orf48 cDNA fragment as a probe confirmed abundant expression of a 4-kb transcript in most of the eight PDAC cell lines we examined, but its expression was hardly detectable in any normal organs except the testis. Moreover, immunohistochemical analysis using anti-C12orf48 antibody showed positive signals in the nuclei of 21 of 31 PDAC tissues, whereas no staining was observed in any of normal pancreatic tissues. MTT assay and colony formation assay revealed that depletion of C12orf48 in KLM-1 and SUIT-2 cells caused dramatic reduction in the number of viable cells. Furthermore, we performed FACS analysis after depletion of C12orf48 by siRNA oligonucleotide in KLM-1 and SUIT-2 cells and found a drastic increase of cells at sub-G1 population. These findings implied its critical roles in pancreatic carcinogenesis.

Importantly, we demonstrated that C12orf48 protein could physically interact with PARP-1(Fig. 1) and positively regulate the enzymatic activity of PARP-1(Fig. 2). We observed that addition of C12orf48 protein significantly enhanced the incorporation of [32P]NAD+ to recombinant PARP-1 protein in vitro when damaged DNA was co-incubated (Fig. 2a). PARP-1 activities to modify histone H1 were also significantly enhanced by overexpression of C12orf48 in HEK293 cells. Furthermore, Concordant with C12orf48 expression, the PARP-1 activities to modify histone H1 were decreased to 40.8% and 34.8% in C12orf48-depleted KLM-1 and SUIT-2 cells, respectively, compared with the control cells (Fig. 2b). The magnitude of this suppressive effect of C12orf48 on PARP-1 activity was almost same as the effect when PARP-1 itself was knocked down (Fig. 2b). It suggested that depletion of C12orf48 could decrease PARP-1 enzymatic activity both in vivo and in vitro. Together, these findings presumably explain that C12orf48 depletion lead to the reduction of pancreatic cancer cell viability, in part, through its direct interaction with PARP-1. However, it cannot be excluded that other C12orf48-specific and PARP1-independent effects can also affect cancer cell viability, and further study is required to clarify the roles of C12orf48 in cancer.

PARP-1 has an emerging and indispensable role in the repair of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). In regard to DNA damage signaling, PARP-1 is promptly stimulated and recruits the enzymes required for DNA repair to the site of DNA damage. Hence, the activity of PARP-1 plays a key role in signaling and initiating these processes. It has been reported that inhibition of PARP-1 activity could increase the susceptibility of cells to DNA damaging agents. Given the findings that C12orf48 could regulate PARP-1 activity, we assessed that the C12orf48 depletion could sensitize cancer cells to various DNA damaging agents. As a result, C12orf48-depleted KLM-1 cells showed much higher sensitivities to adriamycin treatment, UV irradiation, and H2O2 treatment (Fig. 3). These findings indicated that C12orf48 might protect cancer cells from cell death following the DNA damage or cellular stresses in cancer cells through the regulation of poly(ADP-ribosyl)ation activity of PARP-1. It also suggested the possibilities that C12orf48, termed PARP-1 binding protein (PARPBP), might be involved in multiple cellular processes including DNA repair, chromatin modification, cell-cycle progression and genomic stability through the interaction and regulation of PARP-1.

In our studies, we also investigated that knockdown of C12orf48 as well as PARP-1 caused the failure of the G1/S cell-cycle checkpoint which would usually prevent the replication of cells having defects in DNA. Hence, this G1/S checkpoint failure induced by depletion of C12orf48 or PARP-1 in cancer cells could increase a possibility of accumulation of genetic mutations and/or genomic instability, resulting in growth retardation of cancer cells. Moreover, knockdown of C12orf48 in cancer cells enhanced G2/M arrest in PDAC cells after gamma-irradiation, consistent with previous reports describing that PARP-1 inhibitors enhanced the G2 arrest after gamma-irradiation. However, since the underlying mechanism of PARP-1 enzymatic activity in G2-arrest regulation is unclear, additional studies will be required to clarify it.

Together, development of drugs inhibiting the interaction between C12orf48/PARPBP and PARP-1 should be a good therapeutic approach to achieve very specific cytotoxicy to some of pancreatic cancer cells with minimum risk of adverse effects to normal organs.

本論文は1章からなり、膵臓がん新規治療法的分子であるC12orf48の同定とその機能解析について述べられている。膵がんは最も予後の悪い難治性がんの代表格であり、その5年生存率は5%にも満たない。この極めて治療抵抗性である膵がんに対する新規の分子標的を探索する目的で、マイクロアレイによる膵がん細胞の遺伝子発現解析を行っており、このゲノムワイドでの遺伝子発現情報を基に、今回、機能が全く不明であるC12orf48遺伝子が膵がん細胞特異的に高発現していることを見出した。ノーザンブロット解析では、C12orf48の発現はヒトの正常臓器においては、精巣以外には見られなかったことから、C12orf48遺伝子はがんー精巣抗原をコードしているもの考えられる。これらの癌特異的な発現パターンは、自前で作成したポリクローナル抗体を用いた臨床サンプルの免疫組織染色解析でも証明している。

機能の点においては、siRNAでC12orf48の発現を抑制すると、複数の膵がん細胞株の増殖が抑制され、C12orf48は膵がん細胞の増殖や生存の維持にとって、重要な役割を果たしていることが示唆される。免疫沈降法および質量分析解析により、C12orf48蛋白がDNA修復機構の要であるPARP-1蛋白と直接結合することを見出し、免疫沈降法によりそれを証明した。最も興味深い点は、C12orf48蛋白はポリADP-リボースポリメラーゼ-1(PARP-1)と直接結合することにより、その酵素活性を正に制御していることであり、in vitroおよびin vivoの実験にて、この事項を明確に証明している。PARP-1は、現在乳がんなどの分子表標的として幾つかの阻害剤が開発され非常に注目を集めており、C12orf48はPARP-1の酵素機能を活性化させる分子であることを初めて発見しており、大変興味深い。PARP-1は、様々な核蛋白質にポリADPリボースを付加することにより、DNA損傷の感知およびDNA修復の制御を様々な過程において担っている。C12orf48はPARP-1活性を正に制御していることから、C12orf48の発現抑制を行った状態で、抗がん剤や紫外線などによって様々にDNA損傷を与えた場合、膵がん細胞株の生存率がさらに低下した。以上のことから、膵がん細胞特異的に発現しているC12orf48は、PARP-1の活性を正に制御することによって、癌細胞をDNAダメージから保護していることを示唆するものであり、膵がんの化学療法抵抗性の機序の1つを担うものと考えられる。また、C12orf48の機能またはPARP-1との相互作用を抑制することによって、膵がんの化学療法の感受性が増強することが期待され、PARP-1同様、癌の分子標的として有望であると考えられる。

なお本論文は、中川英刀、植田幸嗣、Suyoun Chung、柏谷琴映、江口英利、大東弘明、石川治、醍醐弥太郎、松田浩一、中村祐輔との共同研究であるが、論文提出者が主体となって分析及び検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(生命科学)の学を授与できると認める。