IGFBP3透過調控血管增生與缺氧誘導因子的表現趨勢來抑制卵巢上皮細胞癌的侵襲與惡化

IGFBP3 inhibits the invasion and progression of epithelial ovarian cancer by regulating the expression of angiogenesis and activation of hypoxia-inducible factor

施和均(Ho-Jun Shih)

指導教授 ： 童寶玲

共同指導教授 ： 陳祈玲(Chi-Ling Chen)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200857

摘要

卵巢癌(ovarian cancer)是致命的婦科癌症，多數的患者被診斷出罹患卵巢癌時已經是晚期，即便經手術、藥物治療，但是復發機率高，死亡率亦高。血清類胰島素生長因子結合蛋白-3(IGFBP-3)已被證實具有抑制細胞生長的功能，過去的研究也證實IGFBP3在低侵襲性的卵巢上皮細胞癌(epithelial ovarian cancer, EOC)中有較多的表現，同時IGFBP3有抑制高侵襲性卵巢上皮細胞癌生長的功能。在本研究中，首先找到了一個與IGFBP3有相同表現趨勢、有抑制血管新生功能的蛋白質，血小板反應蛋-1(Thrombospondin-1, THBS1, TSP1)；在EOC 細胞系中恢復IGFBP3的表現後，THBS1的表現也隨之增加。IGFBP3的表現能有效抑制HUVECs的網狀結構生成，也顯著的抑制雞胚胎尿囊絨毛膜 (chick embryo chorioallantoic membrane, CAM)的血管發育；但這些血管生成被抑制的現象在將THBS1剔除之後就被恢復。在小鼠異種移植試驗中發現，有表現IGFBP3的EOC腫瘤其生長會被抑制，同時腫瘤中的血管的生成也被抑制。在啟動子(promoter)活性試驗中發現，THBS1的啟動子在IGFBP3存在時會被激發。實驗結果顯示，IGFBP3可以透過調控THBS1的啟動子來調節THBS1的生成，並且透過THBS1達到抑制血管生長進而減緩腫瘤增殖，但是這個調控作用只是短暫的。腫瘤在增長且沒有足夠的血管提供氧氣時會進入缺氧狀態，接著，腫瘤細胞會活化缺氧誘導因子(hypoxia-inducible factors, HIFs)以調節細胞的代謝並誘導新血管生成以對應缺氧刺激。小鼠實驗中發現，生長被抑制的腫瘤再增生，是腫瘤在缺氧環境下，初期以活化HIF-1α、後期以活化 HIF-2α來達到調節細胞生長、促進新血管的生成，來對應腫瘤的缺氧壓力。過去的研究指出，HIF-1α 和 HIF-2α 都與 IGFBP3 表達有關；本研究透過細胞實驗與異種移植腫瘤的免疫組織染色結果顯示，在高侵襲性卵巢上皮細胞癌中HIF-2α是主要被活化的缺氧誘導因子，而且卵巢癌細胞是活化HIF-1α或 HIF-2α是與細胞的IGFBP3表現量有關聯量。在這個研究中，首先確定IGFBP3主要透過細胞內訊號調控機制來活化THBS1的表現，從而減少腫瘤的血管生成。第二是確定HIF-2α為EOC面對缺氧環境時的主要調節機制。當腫瘤或細胞進入缺氧環境，高侵襲性的EOC細胞傾向活化HIF-2α，HIF-2α對於上皮性卵巢癌從生長抑制狀態轉變為增殖狀態扮演重要調控角色。同時，由於THBS1的表現與IGFBP3呈現正相關，HIF-2α則與IGFBP3有負關聯，這個研究結果未來可能能為卵巢癌的診斷與治療策略提供有效的參考指標。

關鍵字

血清類胰島素生長因子結合蛋白-3 ； 血小板反應蛋白-1 ； 缺氧誘導因子 ； 血管新生 ； 卵巢癌

並列摘要

Ovarian cancer is the most lethal gynecological cancer, and it is frequently diagnosed at advanced stages with recurrences after treatments. Insulin-like growth factor-binding protein-3 (IGFBP3) has been postulated as a mediator of growth suppression signaling. It was shown to function as a suppressor of invasion in epithelial ovarian cancer (EOC). In this study, we identified an angiogenesis inhibitor, thrombospondin-1 (THBS1), which correlated with IGFBP3 expression in EOC cells. After the restoration of IGFBP3 expression in the high invasiveness (with weak IGFBP3 expression) EOC cell line, the transfectants showed an increase in IGFBP3 associated with a parallel increase in THBS1. IGFBP3 decreased cell capillary tube formation in HUVECs, and decreased blood vessel development in chick embryo chorioallantoic membrane (CAM) assay; angiogenic capacity was restored after THBS1 depletion in both assays. Luciferase promoter assay illustrated that the THBS1 promoter was activated in the presence of both intracellular and extracellular IGFBP3. Heterotransplantation of IGFBP3 transfectants significantly decreased tumor growth and vascular formation. Although IGFBP3 can effectively downregulate tumor proliferation and suppress vasculogenesis through THBS1 regulation, its effects are only transient. This is consistent with the model that tumors enter a hypoxic state when they grow large and become less vascular. Then, the tumor cells adapt to hypoxic stress by activating HIFs to regulate cell metabolism, cell proliferation and induce vasculogenesis. When IGFBP3 was transiently expressed in highly invasive ovarian cancer heterotransplants, the xenograft tumors demonstrated a transient growth arrest associated with tumor de-vascularization causing tumor cell hypoxia. Then, tumor re-proliferation was associated with early HIF-1α and later HIF-2α activations. Both HIF-1α and HIF-2α were related to IGFBP3 expressions. Immunostaining and xenograft studies showed that HIF-2α was the major activated HIFs protein when IGFBP-3 was down-regulated. In conclusion, we have identified a novel association between IGFBP3 expression and THBS1 up-regulation, which consequently results in a decrease in angiogenesis. IGFBP3 could activate THBS1 through promoter regulation mainly via an intracellular signaling pathway. Such ability in angiogenesis-regulation could be associated with tumor progression. Furthermore, it seems that the presentation of HIF-2α at tumor hypoxia during tumor devascularization is crucial in switching the cancer cells from a dormancy state to proliferation. Our study demonstrated a novel mechanism for how cancer cells may overcome growth restrictions during hypoxia and maintain their aggressive behavior. These cancer hallmarks in angiogenesis and hypoxia could be utilized as better target therapy in the future.

並列關鍵字

IGFBP3 ； THBS1 ； HIFs ； angiogenesis ； ovarian cancer

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