在黑色素瘤中，BRAF 基因突變最為常見，尤其是 V600E 突變位點，這使得 BRAF 抑制劑在臨床治療上被廣泛應用。儘管標靶治療 (targeted therapies) 與免疫治療在黑色素瘤治療上取得了顯著進展，但多數患者在接受治療數月後，仍會產生對 BRAF 抑制劑的抗藥性。BRAF 抑制劑的抗藥性機制，主要涉及透過基因突變或表現量的改變，使得 MAPK/ERK 或 PI3K/AKT 訊息傳遞路徑 (signaling pathway) 恢復活性。然而，由 BRAFV600E 抑制劑所引發之繼發性腫瘤生成 (secondary tumorigenesis) 的確切生物學機制，目前仍未被完全闡明。此外，過去文獻指出，癌症幹細胞 (cancer stem cells， CSCs) 以及動態的腫瘤微環境 (tumor microenvironment， TME) - 特別是腫瘤相關巨噬細胞極化為具免疫抑制能力的 M2 表現型 (M2-like phenotype) - 在對抗癌治療 (包含 BRAF 抑制劑) 的抗藥性中扮演了重要角色。另一方面，儘管 BRAF 抑制劑對黑色素瘤細胞有效，但矛盾的是，它們同時也會導致繼發性腫瘤的形成。經過一段時間的治療後，這些抑制劑會增強 BRAF 與 CRAF 分子間的結合以形成二聚體 (dimers)，進而刺激並活化 MAPK 路徑，最終導致源自角質細胞 (keratinocytes) 的繼發性腫瘤形成。因此，我們迫切需要開發替代的治療策略，以進一步提升臨床療效。 miR-567 已被證實為包括黑色素瘤在內等多種癌症的腫瘤抑制因子 (tumor suppressor)。 MAPK/ERK 路徑在黑色素瘤的進展中扮演核心角色，我們先前的微陣列分析 (microarray analyses) 研究鑑定出 22 種與該路徑狀態相關的微小 RNA (miRNAs)，其中 miR-567 被視為關鍵的候選因子。此外，我們之前的研究亦顯示，黑色素瘤細胞所釋放的外泌體 (exosomes) 可能促成源自正常角質細胞的繼發性腫瘤形成，並在細胞間通訊 (intercellular communication) 中發揮重要作用。 在本研究中，我們探討了 miR-567 在具 BRAF 抑制劑抗藥性的黑色素瘤、巨噬細胞，以及由 BRAF 抑制劑誘發的繼發性腫瘤中的生物學功能。結果顯示，miR-567 的過度表現 (overexpression) 能顯著降低抗藥性細胞的增殖與遷移能力。此外，miR-567 亦能透過減少親代與抗藥性細胞的成球數量 (sphere formation number) 來影響癌症幹細胞的特性。再者，來自 M2 巨噬細胞與黑色素瘤相關巨噬細胞 (MAMs) 的條件培養基(conditioned media， CM) 會促進黑色素瘤細胞的存活，而 miR-567 的過度表現成功地抑制了這些促進腫瘤生長的效應，包含降低了黑色素瘤細胞的存活率與遷移能力。此外，我們收集了接受 miR-567 模擬物 (mimic) 及 PLX4032 處理後的黑色素瘤細胞條件培養基，並從中分離出外泌體。有趣的是，miR-567 的過度表現能顯著降低角質細胞的遷移活性。最後，我們發現 STAT3 可能是影響黑色素瘤中 miR-567 調控機制的候選轉錄因子 (transcription factor)。 總結來說，miR-567 不僅能減弱具 BRAF 抑制劑抗藥性之黑色素瘤細胞的惡性度，還能透過中和 M2 巨噬細胞誘導的保護作用來破壞有害的腫瘤微環境交相作用 (TME crosstalk)，並減少角質細胞的遷移。此外，STAT3 成為在黑色素瘤進展過程中調控 miR-567 功能的候選轉錄因子。我們提出，miR-567 有望作為治療黑色素瘤的潛力治療標的 (therapeutic target)，以克服微環境介導的抗藥性並預防繼發性腫瘤的發生。;In melanoma， mutations in BRAF are the most common， particularly at position V600E which leads to BRAF inhibitors are widely applied in treatment. Although targeted therapies and immunotherapies have provided significant advancements in melanoma treatment， after several months of treatment， BRAF inhibitor resistance has emerged in most patients. The mechanism of BRAF inhibitor resistance involves recovery of the MAPK/ERK or PI3K/AKT signaling pathway via mutations or changes in expression. However， the exact biological mechanisms driving BRAFV600E inhibitor-induced secondary tumorigenesis remain not fully defined. Besides， it was reported that cancer stem cells (CSCs) and the dynamic tumor microenvironment (TME) - specifically the polarization of tumor-associated macrophages into an immunosuppressive M2-like phenotype play an important role in resistance to cancer therapies including BRAF inhibitor. On the other hand， although BRAF inhibitors are effective against melanoma cells， they paradoxically lead to the formation of secondary tumors. After a period of treatment， they enhance the binding between BRAF and CRAF molecules to form dimers， thereby stimulating activation of the MAPK pathway， resulting in the formation of secondary tumors from keratinocytes. Therefore， an urgent need to develop an alternative strategy should be investigated to further improve clinical benefits. miR-567 has been identified as a tumor suppressor in various cancers， including melanoma. The MAPK/ERK pathway plays a major role in melanoma progression， and our previous study in microarray analyses identified 22 microRNAs (miRNAs) related to the status of this pathway， with miR-567 emerging as a key candidate. Moreover， our previous study showed that the exosome release from melanoma cells may contribute to the formation of secondary tumors which originate from normal keratinocytes， play an important role in intercellular communication. In this study， we investigated the biological functions of miR-567 in BRAF inhibitor-resistant melanoma， macrophages and BRAF-inhibitor-induced secondary tumors. The result show that overexpression of miR-567 could reduce proliferation and migration of resistant cells. In addition， miR-567 could affect cancer stem cell abilities by reducing the sphere formation number of both parental and resistant cells. In addition to， conditioned media (CM) from M2 macrophages and MAMs enhanced the survival of melanoma cells， the overexpression of miR-567 successfully suppressed these tumor-promoting effects in both the survival and migrative abilities of melanoma cells. Besides， we also collected condition media and isolated exosome after melanoma cells were treated with miR-567 mimic and PLX4032. Interestingly， overexpression miR-567 could decrease migration activity on keratinocytes. Finally， we found that STAT3 could be a candidate transcription factor that could influence the regulation of miR-567 in melanoma. In conclusion， miR-567 not only affects the BRAF inhibitor-resistant melanoma cells malignancy but also disrupts harmful TME crosstalk by neutralizing M2 macrophage-induced protection and reduces the migration of keratinocytes. Besides， STAT3 could be a candidate transcription factor that regulates the miR-567 function in melanoma progression. We proposed that miR-567 could act as a potential therapeutic target for treating melanoma， overcoming microenvironment-mediated resistance and preventing secondary tumors.
