Breast cancer is the most common cancer in women worldwide. Incidence of breast cancer is increasing in most countries including the USA and Japan. Development of molecular-targeted therapeutic drugs, such as tamoxifen, aromatase inhibitors, and trastuzumab have contributed to a reduction of mortality rate and improving the quality of life of women diagnosed with breast cancer. However, these drugs have been shown the adverse reactions like the increases in the risk of endometrial cancer with long-term tamoxifen administration and severe cardiac toxicity with trastuzumab treatment. Therefore, it is necessary to search for novel anticancer-drugs with the minimum risk of adverse reactions.

Gene-expression profiles obtained by cDNA microarray analysis have been proven to provide a detailed characterization of individual cancers, and such information should contribute to select more appropriate clinical strategies for individual patients through the development of novel drugs and providing the basis for personalized treatment. Toward such goals, my laboratory had established genome-wide gene expression profiles of 81 breast tumors and 29 normal human tissues by means of cDNA microarray representing 23,040 cDNAs or ESTs, and identified dozens of molecules that were over-expressed in a great majority of breast cancers and were undetectably expressed in normal human organs, especially heart, lung, kidney and liver. Among them, I focused on the identification and characterization of novel genes, brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) and chromosome 12 open reading frame 32 (C12orf32) as therapeutic molecular targets for breast cancer.

<BIG3>

We confirmed up-regulation of BIG3 gene in nine out of 12 clinical breast cancer specimens, compared with normal breast ductal cells as well as mammary gland by semiquantitative RT-PCR. Subsequent northern-blot analysis also confirmed overexpression of its 15-kb transcript in breast cancer cell lines, but its undetectable expression in normal human organs except the brain as concordant to the results of cDNA microarray analysis. To examine a possible biological role of BIG3 in mammary carcinogenesis, we knocked down the expression of endogenous BIG3 using a mammalian vector-based RNA interference technique, and found that knocking-down of BIG3 expression drastically suppressed the growth of breast cancer cell lines, SK-BR-3 and BT-474.

Since the biological functions of BIG3 are totally unknown, we searched for a protein(s) interacting with BIG3 by immunoprecipitation and mass spectrometry analyses, and identified Prohibitin 2/repressor of estrogen receptor activity (PHB2/REA) as a binding partner of BIG3. Subsequent co-immunoprecipitation experiments and immunoblot assays confirmed an interaction of Flag-tagged BIG3 with endogenous PHB2/REA protein in SK-BR-3 cells. Since PHB2/REA was reported to selectively repress the transcriptional activity of ERα through its interaction with ERα in the nucleus, I investigated the direct interaction between BIG3 and ERα, but failed to indicate their interaction.

Because PHB2/REA was reported to be localized mainly at the cytoplasm and to be translocated to the nucleus in ERα-positive breast cancer cells after the estradiol (E2)-treatment, I hypothesized that BIG3 might interact with PHB2/REA in the cytoplasm and interfere with its nuclear-translocation. To examine my hypothesis, I investigated the subcellular distribution of PHB2/REA protein in the presence or absence of BIG3. PHB2/REA was localized in the cytoplasm of MCF-7 cells, in which BIG3 protein was overexpressed, with or without treatment of E2. Moreover, I confirmed that endogenous PHB2/REA was translocated into the nucleus of T47D cells, in which BIG3 was expressed at a very low level after treatment with E2. On the other hand, PHB2/REA remained in the cytoplasm even with E2 treatment when BIG3 was exogenously introduced into T47D cells. Moreover, I confirmed that intracellular-localization of PHB/REA was mostly consistent with the cytoplasmic localization of BIG3 protein in breast cancer tissue by immunohistochemistry. These findings suggest that BIG3 interacted with PHB2/REA and interfered with its nuclear translocation in breast cancer cells.

Furthermore, I investigated the subcellular localization of endogenous PHB2/REA in MCF-7 cells in which endogenous BIG3 expression was knocked down using the siRNA oligonucleotides targeting BIG3. The cell population of nuclear translocated-PHB2/REA was significantly increased in si-BIG3-transfected MCF-7 cells with E2 treatment, compared with those in si-BIG3-transfected MCF-7 cells without E2 treatment. To further examine whether BIG3 protein can enhance the ERα transcriptional activity in breast cancer cells, I performed a reporter assay after knocking-down of BIG3 expression in MCF-7 cells. The depletion of BIG3 expression showed the significant decrease of ERα transcriptional activity. These findings suggest that the presence of BIG3 protein is likely to enhance the ERα transcriptional activity through the inhibition of nuclear translocation of PHB2/REA in breast cancer cells.

<C12orf32>

I confirmed up-regulation of C12orf32 in five out of 11 breast cancer specimens, compared with normal breast ductal cells as well as mammary gland by semi-quantitative RT-PCR. Subsequent northern-blot analyses revealed that two transcripts of C12orf32 (approximately 1.8-kb and 1.5-kb) in breast cancer cell lines were up-regulated, whereas the expression of both transcripts was hardly detectable in normal human organs except testis, prostate, ovary, thymus and small intestine in concordant with the results of cDNA microarray analysis. According to sequencing analysis of these two transcripts, they share the same open reading frame encoding a 238 amino acids peptide, seemed to correspond to the two bands observed in northern-blot analyses.

To assess a growth-promoting role of C12orf32 in breast cancer cells, we knocked down the expression of endogenous C12orf32 in breast cancer cell lines, HBC4 and T47D, which expressed a high-level of C12orf32, by means of the mammalian vector-based RNA interference (RNAi) technique. Knock-down of C12orf32 expression showed significant decrease of cell growth compared with a control si-EGFP-transfected cells. Moreover, I confirmed the results of specificity to its knockdown effects by using mismatched shRNA that contained 4-base replacement in the effective shRNA sequence. Furthermore, I identified an increase in the population of sub-G1 cells in the cells with siRNA-oligonucleotides targeting C12orf32, although no increase of sub-G1 population was observed in those transfected with si-EGFP as a control, indicating that inhibition of C12orf32 expression might induce apoptosis.

To further investigate expression level of endogenous C12orf32 protein in breast cancer cells, I generated a polyclonal antibody to C12orf32 protein (α-C12orf32), and then performed western-blot analysis using cell lysates from seven breast cancer cell lines. Unexpectedly, I observed the smaller size band (16-kDa) in most of breast cancer cell lines examined, although I observed the predicted size of C12orf32 protein (34kDa) in the C12orf32 construct exogenously-expressed-COS7 cells. To investigate whether this smaller band corresponds to the endogenous C12orf32 protein, I knocked down the C12orf32 expression by siRNA in breast cancer cells. I found that the expression of 16-kDa protein was also decreased at protein level as well as transcriptional level, suggesting that this smaller-size protein is corresponding to the endogenous C12orf32 protein in breast cancer cells.

Furthermore, I investigated the subcellular-localization of endogenous C12orf32 protein in breast cancer cells by immunocytochemistry using α-C12orf32. It was observed the cell-cycle-dependent localization of endogenous C12orf32 protein in T47D breast cancer cells. The endogenous C12orf32 was mainly localized in the nucleus of interphase cells, but was observed diffusely from prophase to anaphase. Finally, this protein was concentrated at the contractile ring of cells in telophase. Subsequently, I investigated the effects on each cell-cycle phase by FACS analysis when knocked down of C12orf32 expression in T47D cells by si-C12orf32 after synchronization at the G1/S boundary by aphidicolin treatment. Compared with si-EGFP-treated cells (control), depletion of C12orf32 expression resulted in the increase of cell population of G1 phase, whereas significant reduction of cell population of S phase, suggesting that depletion of C12orf32 might induce the inhibition of G1/S transition. These results suggest that C12orf32 might have an important role to G1/S transition.

In conclusion, the results of my study about BIG3 and C12orf32 clearly suggest that BIG3 and C12orf32 are specifically and frequently overexpressed in breast cancers, and downregulation of these two molecules by treatment with shRNA significantly suppresses the growth of breast cancer cells, indicating their crucial role in the growth of breast cancer cells. Taken together, these findings should contribute to a better understanding of mammary carcinogenesis, and imply that BIG3 and C12orf32 are promising molecular targets for breast cancer treatment.

現在、乳癌は本邦女性の悪性腫瘍罹患率1位であり、死亡者数も増加の一途を辿っている。乳癌の治療としては、タモキシフェンやアロマターゼ阻害剤が再発のリスクを軽減するための術後補助療法や進行・再発乳癌の標準治療法として利用されており、顕著に生存率を向上させてきた。さらに、乳癌症例の約30%に増幅の認められるHER2を標的とした抗体医薬トラスツズマブなどの分子標的薬剤も開発されており、生存率の向上に加えて患者の生活の質も改善されてきた。しかしながら、タモキシフェンの長期投与による子宮内膜癌のリスクの上昇、アロマターゼ阻害剤による骨量減少やトラスツズマブの心毒性といった重篤な副作用もあり、より副作用の少ない新規分子標的治療薬の開発が切望されている。

本研究は、乳癌の新規治療薬の開発およびその発症機構の解明を目的とし、cDNAマイクロアレイ法による乳癌・ヒト正常臓器における遺伝子発現解析を通じて、乳癌において高頻度に発現亢進を認め、ヒト正常臓器では発現の低い2遺伝子(brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3)): chromosome 12 open reading frame 32 (C12orf32))を抽出し、それらの機能解析を行ったものである。

(BIG3)

1.乳癌臨床検体由来mRNAを用いた半定量的RT-PCRの結果、BIG3遺伝子は12例中9例にて高レベルの発現を認めたが、ヒト正常臓器由来mRNAを用いたノザン解析では、発現は認めなかった。さらにBIG3特異的ポリクローナル抗体を用いた免疫組織染色では、乳癌組織のみで染色を認め、BIG3が乳癌特異的分子であることがわかった。

2.BIG3の乳癌細胞増殖への関与を調べるために、BIG3の高レベルの発現を認める乳癌細胞株(SK-BR-3およびBT-474)にBIG3遺伝子特異的RNA干渉(shRNA)発現ベクターを導入し、この遺伝子発現を抑制した結果、顕著な細胞増殖抑制効果が認められた。以上より、BIG3は乳癌細胞増殖に重要な役割を果たすことが示唆された。

3.BIG3の乳癌細胞増殖における機能を探るために、BIG3結合蛋白質の探索を行った。BIG3発現ベクターを乳癌細胞株BT474細胞に発現導入し、免疫沈降法および質量分析法を行った結果、ER選択的調節因子Prohibitin 2/repressor of estrogen receptor activity (PHB2/REA)を同定した。次に、BIG3とPHB2/REAの発現ベクターを用いた免疫沈降法および免疫細胞染色により、乳癌細胞での両蛋白質の結合と細胞質での共局在を確認した。

4.PHB2/REAはこれまでにエストロゲン(E2)存在下で細胞質から核移行してエストロゲン受容体(ERα)と結合し、その転写活性を抑制することが報告されている(PNAS.1999;96:6947)。この事実を踏まえて、以下の実験を試みた。ERα陽性・BIG3高発現乳癌細胞株MCF7・ZR75-1にBIG3特異的siRNAを導入してBIG3遺伝子発現を抑制したところ、E2存在下においてPHB2/REAの核内移行が確認された。一方、コントロールsiRNA(siEGFP)を導入した細胞では、E2存在下にもかかわらず細胞質に局在が認められた。また、その際のレポーターアッセイでは、BIG3発現抑制によるERα転写活性抑制も確認された。以上より、BIG3は細胞質内にてPHB2/REAと結合することによりE2依存的PHB2/REAの核移行を阻害し、PHB2/REAのER転写活性化抑制能を阻害することでERαの恒常的活性化を導くことを証明した。

(C12orf32)

1.乳癌臨床検体より抽出したmRNAを用いた半定量的RT-PCRの結果、C12orf32遺伝子は11例中5例にて高発現を認めた。ノザン解析では、精巣、前立腺、卵巣、胸腺、小腸において弱い発現を認める一方、他の正常臓器では認められなかった。

2.C12orf32の乳癌細胞増殖への関与を調べるために、C12orf32の高い発現を認める乳癌細胞株(HBC4およびT47D)にC12orf32遺伝子特異的shRNAを導入し、この遺伝子発現を抑制した結果、顕著な細胞増殖抑制効果が認められた。またフローサイトメトリー解析よりsubG1細胞の増加が確認されたことから、アポトーシス誘導が示唆された。以上より、C12orf32も乳癌細胞増殖に必須であることが示唆された。

3.C12orf32特異的ポリクローナル抗体を作製し、ウェスタン解析を行ったところ、ほとんどの乳癌細胞において内在性C12orf32は、予測分子量(34KDa)よりも小さい(約16kDa)ことがわかった。さらに内在性C12orf32の細胞内局在を調べたところ、間期では核内に局在し、分裂前期から後期にかけては細胞全体一様に染色を認め、分裂終期では収縮環に局在することがわかった。また各細胞周期における発現を調べた結果、M期に最も強い発現を認めた。以上、C12orf32は乳癌細胞では細胞周期依存的な発現を示すことがわかった。

4.C12orf32の細胞周期における役割を検討するために、C12orf32遺伝子発現抑制による細胞周期への影響を調べた。その結果、G1期細胞の顕著な増加およびS期細胞の減少が認められた。以上より、C12orf32はG1-S期移行に重要であることが示唆された。

本学位論文は、ゲノムワイドな遺伝子発現情報解析を通じて乳癌特異的遺伝子BIG3およびC12orf32を同定し、それらの機能解析を行ったものである。乳癌治療を考える上では、両遺伝子ともに乳癌特異的で細胞増殖に必須であることから、これらを標的とした抗癌剤の開発は副作用の極めて少ないことが期待される。以上より、本研究は、乳癌新規分子標的治療薬の開発および乳癌発症機構の解明に重要な貢献をなすと考えられる。

なお、本論文は、論文提出者が主体となって分析および検証をおこなったもので、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)の学位を授与できると認める。