SOX9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family, is required for the development and differentiation of multiple cell lineages, and has been implicated in several types of cancer; however, there are few reports of SOX9 expression in gastrointestinal cancers. To clarify the significance of SOX9 in gastric carcinoma (GC), immunohistochemical staining of SOX9 and the CpG-island methylation status of SOX9 were evaluated and compared with clinicopathologicalfactors. It was concluded that upregulation of SOX9 is related to GC development. On the other hand, downregulation of SOX9 is associated with GC progression.

The increase of SOX9 occurred from the normal-metaplasia-adenoma transition and SOX9 expression was correlated with CDX2 expression, which is known to play a role in intestinal metaplasia. SOX9 was strongly expressed in 83% (20/24) of gastric adenoma, in which Ki67 was also expressed. Furthermore, MTT assay revealed that SOX9 promoted normal gastric epithelial cell (GES-1) proliferation. Moreover, PI3K/Akt activation was induced by overexpression of SOX9 in GES-1 cells. SOX9 was frequently expressed in the early stage gastric cancer. Correlation between the expression of SOX9 and clinicopathological factors was more significant in the intestinal type than in the diffuse type. The findings underline a novel oncogenic function of SOX9 in the early stage of intestinal type gastric carcinogenesis through regulating PI3K/Akt.

SOX9 function in GC cell lines was then studied. In vitro, MKN7 gastric cancer cells transfected with SOX9 siRNA proliferate more rapidly than control cells by MTT assay. It was also examined whether SOX9 could inhibit Epithelial-Mesenchymal Transition (EMT) in gastric cancer. Knockdown of SOX9 in MKN74 cells induced conversion of circular-shaped cells with cell-cell contact to spindle-shaped cells and improved cell migration, shown by the wound healing assay. MKN7 and NUGC3 cells treated with SOX9 siRNA also induced cell migration. Next, the effect of SOX9 on the expression of EMT marker mRNA and proteins was examined by real-time PCR and Western blotting. Knockdown of SOX9 in MKN74 cells decreased the expression level of E-cadherin, and increased SLUG, ZEB1, and Vimentin expression, which are known as EMT. Comparable results were observed in MKN7 and NUGC3 cells transfected with SOX9 siRNA.

To further study the role of SOX9 in EMT, SOX9 was transfected into GES-1 cells transiently and stably. SLUG levels were significantly reduced by overexpression of SOX9, concomitant with re-expression of E-cadherin. The relationship between E-cadherin and SOX9 in human gastric cancer tissues was further investigated, and a positive correlation was elucidated between SOX9 and E-cadherin. SOX9 may inhibit EMT in the progression of gastric cancer by downregulating SLUG and thereby upregulating E-cadherin. Taken together, these results suggest that SOX9 may contribute to the development of gastric cancer by promoting cell proliferation; on the other hand, SOX9 downregulation induces EMT during progression of GC.

The expression of SOX9 was decreased in 43% of GC tissues. SOX9 methylation was detected in 48% of GCs and correlated with the low expression of SOX9 protein. The decrease of SOX9 expression in advanced GC was related to the methylation of SOX9 promotor during GC progression. An interesting finding in this study was that the expression of SOX9 protein was frequently lost or down regulated in the EBV-positive GC cases. SOX9 expression was significantly downregulated in 4 GC cell lines when infected with EBV (MKN1-EBV, MKN7-EBV, TMK1-EBV and NUGC-3-EBV), and one original EBV-positive GC cell lines, KT. Downregulation of SOX9 expression may be associated with the development of EBV-associated GC. More SOX9 promotor methylation in gastric cancer samples from patients with EBV infection (86%, 6/7) was detected than in those without EBV infection (46%, 52/114, P = 0.041). Furthermore, 68% (5/8) of EBV-infected GC cell lines (MKN1-EBV, MKN7-EBV, TMK1-EBV, NUGC3-EBV and KT) that show relatively low expression were detected SOX9 methylation. These findings might support that SOX9 methylation was accelerated by EBV infection and the subsequent silencing of SOX9 expression may correlate with the progression of gastric cancer.

In summary, SOX9 expression was increased in the development of GC and decreased throughout its progression. The increase might be related to intestinal-type gastric carcinogenesis and influence the proliferation of GC cells via PI3K/Akt. Downregulation of SOX9 seems to contribute to the progression of GC by regulating EMT. Methylation of SOX9 promoter increased during progression and could be the cause of SOX9 suppression in advanced cancer. SOX9 promotor methylation was also related to EBV-associated GC, which shows low expression of SOX9. SOX9 is closely related to gastric cancer.

本研究では、手術検体を用い免疫組織化学的に胃癌におけるSOX9の発現の意味を検討している。さらに胃癌細胞株、EBV感染胃癌細胞株でのSOX9の発現、およびその発現制御機構としてSOX9プロモーターのDNAメチル化状態について検討を加えた。最後に胃正常上皮細胞株および胃癌細胞株を用い、SOX9遺伝子やSOX9 siRNAを導入し、細胞増殖能、運動能に与える影響を調べた。下記の結果を得ている。

1.EBV陰性胃癌においては、SOX9は胃腺腫、粘膜内癌で発現が増加するが、胃癌の進行とともに発現が低下した。このSOX9発現低下は主にSOX9プロモータのDNAメチル化によるものであった。

2.胃正常上皮細胞にSOX9を遺伝子導入することにより増殖率が増加したことから、SOX9は胃癌発生に寄与することが示唆された。又、SOX9による胃正常上皮細胞の増殖率の増加にはPI3K/Akt経路の活性化の関与が示唆された。

3.胃癌細胞株においてSOX9の発現をsiRNAにより抑制すると、細胞形態が上皮様から紡錘形に変化し、細胞運動能の亢進が認められた。また、SOX9 siRNAにより上皮マーカーであるE-cadherinやβ-cateninの発現が低下し、間葉系マーカーであるVimentinの発現亢進が認められた。この結果によりSOX9発現低下はEMT(上皮間葉転換)を誘導することが示唆された。この結果は、胃癌手術検体においてSOX9が発現低下し、E-cadherinの発現が低下していたことと対応している。

4.EBV関連胃癌においては、粘膜内癌の段階からSOX9はプロモーター領域のDNAメチル化により発現低下していた。EBV感染の有無により、SOX9の発癌における役割が異なっている可能性が示された。

本研究では胃癌の進行に伴いSOX9発現が低下することを明らかにし、この発現低下は主にSOX9のプロモーター領域のDNAメチル化によるものであることを見いだした。また胃癌培養細胞の結果においてSOX9発現低下によりEMTが誘導されたことから、胃癌の進行にSOX9の発現低下が関与する事が示唆された。SOX9が胃癌の発癌と進行において異なる役割を果たすことが明らかとなり、さらなる胃癌の進展の機構の解明に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。