HNRNPLL (heterogeneous nuclear ribonucleoprotein L-like) , a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for an approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was decreased upon EMT while those of SP1 and SP3 were unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.

PURPOSE: This study aimed to determine the correlation between DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) status and the response to streptozocin in advanced well-differentiated pancreatic neuroendocrine tumors (WD panNETs). METHODS: To test the hypothesis that MGMT deficiency was required for an alkylating drug response, we retrospectively reviewed the response of 13 patients with WD panNETs to alkylating agents in relation to MGMT status. We also studied MGMT expression in streptozocin resistance using panNET cell lines. RESULTS: The cohort included 54% of patients with and 46% without MGMT expression. Among these, 83.3% (5/6) of MGMT-negative cases showed a partial response to streptozocin. In contrast, only 14.2% (1/7) of MGMT-positive cases showed a partial response (P = 0.013). Induced expression of MGMT in BON1 cells (a panNET cell line with undetectable endogenous MGMT) produced streptozocin resistance. Knockdown of MGMT in QGP1 cells, which express MGMT endogenously, did not alter the response to streptozocin. CONCLUSIONS: We observed a relationship between MGMT status and streptozocin response in both patients and cell culture. Despite limited cases examined, high concordance of negative expression of MGMT and response to streptozocin treatment suggest that MGMT expression can be a potential biomarker for this treatment.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), an RNA-binding protein that regulates alternative splicing of pre-mRNA, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here, we show that HNRNPLL promotes cell cycle progression and, hence, proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed threefold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3 and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and at protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation-promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 h of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3 and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3 and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNA encoding regulators of DNA replication and promotes colorectal cancer cell proliferation.

前年度に引き続き、肺腺がん細胞における一次線毛発現制御分子KATNAL2の機能解明を試みた。方針としては、以下に示す2つの検討を並行しておこなった。 1. 細胞レベルの検討 前年度までの成果から、一次線毛陽性の肺腺がん細胞は高頻度に細胞周期の停止を認めることが既にわかっている。本年度は、この細胞周期停止のメカニズムを一次線毛との関連からアプローチして解明を試みると同時に、昨年度までに樹立したKATNAL2のノックアウト細胞を用いて、抗がん剤の感受性と一次線毛/KATNAL2の関係を検討した。結果、一次線毛陽性細胞は予想外のシグナル伝達経路を介して細胞周期の停止に至っていることが示唆された。このシグナル伝達系の活性化には、腫瘍細胞自体もしくは腫瘍微小環境の関与、いずれの可能性も考えられた。この点は今後臨床検体も用いて詳しく検討する必要がある。一方で、一次線毛/KATNAL2が一部の抗がん剤に対する感受性に大きな影響を与えていることが示唆された。抗がん剤耐性に一次線毛/KATNAL2が関与している可能性を示す結果であるが、一次線毛陽性細胞がいわゆるがん幹細胞と必ずしも一致するわけではないようである。この点も今後詳細な検証を要する。 2. 生体レベルの検討 国立長寿医療センター研究所との共同研究により、KATNAL2のコンディショナルノックアウトマウスの作出を試みている。昨年度は、マイクロインジェクション装置に故障が発生し、その更新に時間を要したため、目的のマウスを得るには至らなかった。

低酸素によって同時に転写誘導される一連の細胞接着分子遺伝子の協同作用を解析した。またこれら以外に低酸素によって転写誘導されることがこれまで知られていなかった細胞接着分子があるかどうかについても検索した。その結果、低酸素によって糖脂質の糖部分と脂質部分に並行して変化が誘導され、糖蛋白質においても蛋白や糖側鎖に並行して変化が誘導され、これらの並行的変化が細胞の低酸素応答において互いに相乗的に機能することを示唆する成績が得られた。

細胞接着に関与する遺伝子のうちに、低酸素誘導因子(HIF)により誘導されるものを検索し、多数の遺伝子を見いだした。これらの遺伝子のHIFによる誘導機構を解析し、一部の遺伝子群は調節領域に複数の低酸素反応エレメント(HRE, hypoxia-responsive-element)を持ち、HIF単独で有意な転写誘導が見られること、他の一群の遺伝子は単一のHREを持ち、HIF以外の転写因子と協同して転写を誘導することを明らかにした。後者については共役転写因子の同定を行った。