【Introduction】

Colorectal cancer is one of the most common malignancies and remains main causes of cancer-related deaths due to therapeutic resistance. Although molecular mechanisms involved in development or progression of colorectal cancer have been studied for a long time, they haven't been still fully understood. Therefore, it is necessary to find new insights in the process of colorectal cancer development or malignant transformation to improve its treatments or diagnostics. In this study, I focused on the factors whose functions are involved in colorectal cancer tumorigenesis.

Chapter 1

【Background】

Sporadic and familial colorectal tumors harbor biallelic adenomatous polyposis coli (APC) mutations, resulting in truncated APC gene products and constitutive activation of Wnt signaling through accumulation of nuclear b-catenin, a major component of its signaling pathway. Although it is widely accepted that the ability of APC to negatively regulate Wnt signaling is essential for its tumor suppressor function, mutations of APC are considered to have other functions in colorectal tumor formation. We have previously identified novel APC binding protein, which have GEF activities specific for Rac1 and Cdc42, named Asef (APC-stimulated guanine nucleotide exchange factor) and Asef2 (Asefs). APC-Asef or Asef2 complex promotes cell migration. Furthermore, we have shown that truncated mutant APCs in colorectal cancer cells (CRCs) constitutively activate Asef and Asef2 and thereby induce aberrant cell migration.

【Results】

Roles of Asefs in intestinal adenoma formation

At first, I examined Asefs expression in human colorectal cancer tissues. Immunohistochemical (IHC) and qRT-PCR analyses showed that both Asef and Asef2 are highly expressed in cancerous tissues compared with the corresponding non-cancerous tissues. Next, to investigate whether Asefs are involved in tumorigenesis in vivo, we generated Asef-/-Asef2-/- mice. These mice seemed to be morphologically normal and were fertile. Then I crossed them with ApcMin/+ mice which have germline mutation in Apc gene and revealed that homozygous Asef and Asef2 deficiency significantly reduced the number and size of intestinal adenomas.

Asefs induce MMP9 expression via JNK pathway

I attempted to examine the mechanisms by which Asefs contribute to intestinal adenoma formation. Activation of Rac1 and Cdc42 have been reported to induce the expression of Matrix Metalloproteinase9 (MMP9) through activation the c-Jun N-terminal kinase (JNK). By using shRNA, dominant negative mutant of Asef and an inhibitor of JNK in colon cancer cell lines, I found that Asef and Asef2 activated by mutant APCs upregulate the expression of MMP9 through activation of JNK signaling. MMP9 is well-known to be crucial for late-stage tumor invasion and metastasis, but also important for the development of benign lesions with its degradation activity against extracellular matrix. Consistent with the data with cell lines, I also showed that Asef and Asef2 regulate phosphorylation of JNK and thereby upregulation of MMP9 expression in intestinal adenomas of ApcMin/+ mice and treatment of ApcMin/+ mice with MMP9 inhibitor suppressed intestinal adenoma formation. Furthermore, I revealed that knockdown of Asefs or APC lead to suppression of invasive activity in colorectal cancer cells only with APC mutations. Taken together, these data suggest that Asefs-mediated upregulation of MMP9 through JNK signaling may contribute to not only intestinal tumorigenesis but also tumor progression.

Crucial functions of Asefs in tumor angiogenesis

Angiogenesis is known to play important roles in the development of intestinal adenomas. In this regard, we previously found that Asef is involved in growth factors induced microvessel formation and tumor angiogenesis. Thus I examined the density of microvessel in adenomas and found that angiogenesis in adenomas from Asef-/-Asef2-/-ApcMin/+ mice was markedly lower than that from ApcMin/+ mice. This result indicates that the growth of adenomas might be retarded, at least in part, due to the impairment of tumor angiogenesis caused by Asefs deficiency.

【Conclusions】

The present study demonstrates that Asefs have critical roles in intestinal adenoma formation (Fig1). I observed that Asef and Asef2 can induce MMP9 expression through activation of JNK pathway and promote invasive activity of CRCs with APC mutations. Furthermore, I showed that Asef and Asef2 are required for tumor angiogenesis. These results implicated Asefs might function both in cancer cells and normal cells. Since Asef and Asef2-deficient mice appear normal and have a lifespan comparable with that of wild type mice, I speculate that compounds targeting Asef family proteins might hold promise as new anti-tumor reagents.

Chapter 2

【Background】

Epithelial tissues of colon and small intestine are one of the most rapidly renewing tissues and the renewing system is sustained by the existence of intestinal stem cells. Recently, it was reported that Leucine-rich repeat containing G-protein coupled Receptor 5 (LGR5) markers colon and intestinal stem cells. LGR5 is an orphan receptor and known to be a target of Wnt signaling. Importantly, LGR5 positive stem cells are cell of origin of intestinal adenomas. Therefore, characterization of LGR5+ stem cells or investigations of factors important for the function of those stem cells have been the focus of intense research interest. Although the importance of LGR5 as a stem cell specific marker is apparent, functions of LGR5 in colorectal tumor formation remains unclear.

【Results】

GATA6 regulates the expression of LGR5 in CRCs

I investigated the role of LGR5 in the tumorigenicity of CRCs and found that knockdown of LGR5 suppresses growth of CRCs in vivo (Fig2). Thus, in order to elucidate the mechanisms underlying the regulation of LGR5 expression in CRCs, I performed an siRNA screen to identify genes involved in the regulation of LGR5 expression in CRCs. As a result of gene screen, I found that knockdown of GATA6, which is a zinc finger transcription factor and essential for embryogenesis, resulted in the significant reduction in LGR5 expression. Luciferase and chromatin immunoprecipitation assays showed that GATA6 regulates transcription of LGR5 through binding to the LGR5 promoter. Moreover, IHC analysis revealed that GATA6 is expressed in intestinal stem cells expressing LGR5.

GATA6 is important for tumorigenicity of CRCs

Although several studies have explored the functional roles of GATA6 in CRCs, it remains unclear whether GATA6 contributes to tumorigenic capacity of CRCs in vivo. Like LGR5, knockdown of GATA6 by lentiviral shRNA significantly reduced the growth of CRCs in nude mice. Interestingly, knockdown of either LGR5 or GATA6 did not affect the growth of CRCs in vitro adherent culture condition but caused the significant reduction in colony-forming ability in soft agar.

The expression of GATA6 is regulated by microRNA-375

Consistent with previous studies, I found that the expression of LGR5 was much higher in human colorectal tumors than in the adjacent normal tissues. Intriguingly, qRT-PCR, immunoblotting and IHC analyses revealed that GATA6 protein but not mRNA levels were upregulated in cancerous tissues. These data implicated that posttranscriptional or posttranslational mechanisms might be involved in the regulation of GATA6 expression in CRCs. As emerging evidence has recently shown important roles of miRNAs in the posttranscriptional regulation, I investigated whether miRNAs regulate GATA6 expression. Among miRNAs that were predicted to target the 3' untranslated region (UTR) of GATA6, miR-375 was found to have the ability to suppress GATA6 expression. Furthermore, the expression of miR-375 was found to be significantly reduced in most colorectal cancer tissues. When transplanted into nude mice, the growth of CRCs overexpressing miR375 was markedly retarded compared with that of parental CRCs. In addition, CRCs overexpressing miR375 showed reduced colony forming ability in soft agar.

【Conclusions】

In this study, I showed that GATA6 upregulates the expression of LGR5 in CRCs and that LGR5 and GATA6 are important for the anchorage-independent growth thereby the tumorigenicity of CRCs. Moreover, it is suggested that downregulation of miR375 is responsible for increased expression of GATA6 in CRCs. Further investigations are needed to determine the ligand or downstream signaling pathway of LGR5 which might be important for tumorigenicity of CRCs. The results presented here suggest that the microRNA-GATA6-LGR5 pathway could be promising molecular targets for therapy of colorectal cancer.

Fig1 Functional roles of Asefs in CRCs

Fig2 LGR5 is required for tumorigenesis of CRCs

Fig3 Tumors dereived from DLD-1 cells infected with control or shRNA-GATA6 expressing lentivirus

癌抑制遺伝子APCは家族性大腸癌の原因遺伝子であり、散発性の大腸癌においても70%以上で変異が見出されている。APCの変異はWntシグナルの亢進を引き起こすことで、大腸癌発症の初期段階に重要であることが知られている。APCはRac1及びCdc42特異的なGEFであるAsefファミリー(AsefとAsef2:以下Asefs)と結合し、そのGEF活性を正に制御することで上皮細胞や血管内皮細胞の運動を制御している。また、大腸癌細胞では変異APCがAsefsを恒常的に活性化し、異常な運動能を誘引していることが分かっている。しかし、実際の大腸癌の発症におけるAsefsの重要性に関しては不明であった。そこで本研究の第1章ではAPCの変異による腸管での腫瘍形成におけるAsefs生理的意義を明らかにすることを目的として解析を行った。

APCに変異を持つ大腸癌のモデルマウス(Apc(Min/+))とAsef(-/-)Asef2(-/-)マウスをかけあわせることで、Asefsの欠損したApc(Min/+)マウスを作製した。その結果、Asefsの欠損によってApc(Min/+)マウスの腸管で発生する腫瘍の数が顕著に減少したことから、APCの変異による腫瘍形成にAsefsが重要な機能を担っていることが明らかとなった。

次に腫瘍形成においてAsefsが担っている機能を分子レベルで明らかにするため、大腸癌細胞を用いたin vitroの解析を行った結果、Asefsは変異APCによって活性化されることでJNKシグナルを活性化し、癌の浸潤・転移に関わるMMP-9の発現を誘導することを見出した。さらに、AsefsによるMMP-9の発現亢進が大腸癌細胞の浸潤を促進することが明らかとなった。そこで、腸管での腫瘍形成におけるMMP-9の生理的重要性を検証するため、ApcMin/+マウスにMMP-9阻害剤を腹腔投与した結果、腫瘍形成の抑制が確認された。また、Asefsを欠損させたApcMin/+マウスの腫瘍では血管新生が抑制されていることを見出した。以上の結果より、MMP-9の発現や腫瘍血管新生におけるAsefsの機能はAPCの変異による腫瘍形成メカニズムの一端を担っていると考えられた。

第1章ではAPCの変異による腫瘍形成におけるAsefsの機能解析を行ったが、近年大腸癌の発症にはLGR5陽性の腸管幹細胞におけるAPCの変異が必要であることが明らかとなってきた。LGR5は大腸癌で発現が亢進している7回膜貫通型タンパク質であることが知られていたが、リガンドや下流のシグナルは未知であった。LGR5をshRNAにより安定的に発現抑制した大腸癌細胞株を樹立したところ、腫瘍形成の顕著な低下が観察された。したがってLGR5は腫瘍形成の起源となる腸管幹細胞のマーカーとしてだけでなく、機能的にも大腸癌細胞の腫瘍形成能に関与していると考えられた。そこで第2章ではsiRNAライブラリーを用いたスクリーニングにより、LGR5の上流で大腸癌細胞の腫瘍形成能に関与する因子を探索し、その機能解析を行った。

スクリーニングの結果より、LGR5の転写を直接制御する転写因子GATA6を同定した。GATA6は腸管幹細胞においても発現が確認されたことから、大腸癌細胞だけでなく正常な幹細胞においてもLGR5の発現を制御している可能性が考えられた。また、GATA6の発現はLGR5と同様に大腸癌で上昇しており、その発現を抑制することで大腸癌細胞の腫瘍形成能は低下した。上述した結果は、GATA6によるLGR5の発現上昇が大腸癌細胞の腫瘍形成能に深く関わっていることを示している。さらに、microRNAによるGATA6の発現調節の可能性についても明らかにした。

以上に示した本研究では、癌抑制遺伝子APCの変異による腫瘍発生における新たな分子メカニズムをin vivoおよびin vitroにおける解析を行うことで明らかにし、また腸管幹細胞のマーカーとして知られていたLGR5が大腸癌細胞の腫瘍形成に機能的に関与していることを示し、さらにその上流における発現調節のメカニズムの一端を明らかにすることができた。また、本研究によって得られた知見は、AsefsやmicroRNA、またLGR5といった分子が大腸癌に対する抗癌剤の新たな標的因子になりうる可能性を示している点において、臨床的にも重要な研究成果だといえる。なお、本論文は川崎善博、室谷研、古川史織、秋山徹との共同研究であるが、いずれも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。よって審査委員一同は、本論文が博士(農学)の単位論文として価値あるものと認めた。