TWI827190B - 標靶治療組合物及其於抑制乳癌細胞增生、移行或侵入之用途 - Google Patents
標靶治療組合物及其於抑制乳癌細胞增生、移行或侵入之用途 Download PDFInfo
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Abstract
本發明揭示一種標靶治療組合物於抑制乳癌細胞增生、移行或侵入之用途,其包括一第3型表皮細胞膜蛋白質靶向抑制劑;該些乳癌細胞具有基因表現上調控之第3型表皮細胞膜蛋白質、上皮細胞成長因子受體(EGFR)基因、第2型表皮細胞生長因子受體 (HER2) 基因、第3型上皮細胞成長因子受體 (HER3)基因、 荷爾蒙受體 (HR)基因,或基因表現下調控之第4型上皮細胞成長因子受體(HER4)基因,在該些乳癌細胞投予具有第3型表皮細胞膜蛋白質靶向性之寡核苷酸或核酸酶,以破壞編碼有第3型表皮細胞膜蛋白質之DNA或mRNA,達到抑制乳癌細胞增生、移行或侵入之效果。
Description
本發明關於一種組合物,尤其關於用於標靶治療之組合物,其對於人類乳癌細胞的增生、移行或侵入具有特異性。
乳癌為世界上最為廣泛確診之癌症,且占了2020年罹癌死亡案例的6.9%(共684,996例)。越來越多證據表明,乳癌不論於分子、病理組織及臨床層次均涵蓋了多種異質子群(heterogeneous subgroup)。乳癌主要有三種亞型腫瘤,其分類係依據雌激素受體(estrogen receptor,ER)、孕激素受體(progesterone receptor,PR)是否表現,及/或第2型表皮細胞生長因子受體(human epidermal growth factor receptor 2,HER2)之表現是否有增幅之情形而定;其中,荷爾蒙受體(hormone receptor,HR)陽性、第2型表皮細胞生長因子受體陰性、第2型表皮細胞生長因子受體陽性及三陰性腫瘤(亦即缺乏三種標準分子標記)分別佔有70%、15至20%及15%之乳癌病患。
當前技術中,主要採納局部處理例如外科手術及放射治療,或系統性治療包括化學治療、荷爾蒙治療及標靶治療。近年來,在醫學上的進步已
逐漸演進至多元模式治療(multimodal therapy),例如輔助免疫療法(adjunct immunotherapy),以提升病患預後品質。
在制定乳癌病患的治療方針之前,必須將腫瘤亞型、解剖癌症分期及病患體質等要件納入評估;舉例來說,荷爾蒙療法係針對雌激素受體陽性或孕激素受體陽性乳癌的主要系統性療法;第2型表皮細胞生長因子受體標靶治療則針對第2型表皮細胞生長因子受體陽性乳癌患者所設計,其利用單株抗體、抗體藥物偶聯物(antibody-drug conjugate)或酪氨酸激酶抑制劑以達到治療效果,但該些病患通常有預後不佳的問題,同時也可能產生化療抗藥性;儘管如此,不論該些病患處於病程早期或晚期,第2型表皮細胞生長因子受體標靶治療仍大幅度的改善了第2型表皮細胞生長因子受體陽性乳癌病患的存活率。
然而,即使標靶藥物有效的提升了乳癌病患的存活率,其潛在的抗藥性仍為難以排除的隱憂;此外,第2型表皮細胞生長因子受體及雌激素受體訊息傳遞路徑之間的串擾(cross-talk)更可能導致內分泌抗性,這有賴對於曲妥珠單抗(trastuzumab)抗性背後機轉的瞭解及新式療法的開發。
表皮細胞膜蛋白質(epithelial membrane proteins,EMPs)屬於生長停滯特異3基因家族(growth arrest-specific 3 gene family),並在細胞遷移、細胞生長及細胞分化等生物功能中扮演著重要的角色;第3型表皮細胞膜蛋白質(EMP3)為一種4-穿膜醣蛋白,隸屬於EMP家族,在人類癌症病理學領域中越來越受關注;第3型表皮細胞膜蛋白質的信使核糖核酸(mRNA)表現量在乳癌細胞當中顯著地高於正常細胞;此外,第3型表皮細胞膜蛋白質的表現與乳腺腫瘤細胞株的侵入性表徵亦有所關聯。
研究指出,將乳腺腫瘤細胞株SKBR3進行第3型表皮細胞膜蛋白質基因減弱,能有效的抑制離體腫瘤的增生,指出了第3型表皮細胞膜蛋白質可能為人類乳癌的致癌基因(oncogene);此外,第3型表皮細胞膜蛋白質的上調控(up-regulation)不僅伴隨了上皮-間質細胞轉化(epithelial-mesenchymal transition),且同時為EMT的離體特徵基因,而當前已分類出6種癌症類幹細胞株(cancer stem cell-like cell lines),其屬於基礎B型並過量表現第3型表皮細胞膜蛋白質。
另一項指出第3型表皮細胞膜蛋白質為人類乳癌致癌基因的證據來自微陣列分析(microarray analysis),其係針對第2型表皮細胞生長因子受體過量表現之永生化管腔人類乳腺上皮細胞(luminal human mammary epithelial cell)進行分析,指出了第3型表皮細胞膜蛋白質不僅僅過量表現,更與第2型表皮細胞生長因子受體表現量具有劑量依賴關係,然其過量表現的臨床意義迄今仍未定論。
為解決上述問題,離體實驗包括正轉染與負轉染將實施於適當的乳癌細胞株以證實本申請人的假設。接著,建立SCID模型以檢驗第3型表皮細胞膜蛋白質作為體內標靶的治療效益。釐清第3型表皮細胞膜蛋白質的潛在臨床意義並作為新的人類乳癌治療標的乃勢在必行。此外,於第2型表皮細胞生長因子受體及第3型表皮細胞膜蛋白質之間所具有的功能性串擾也為本申請人所揭露;在原發乳癌細胞中過量表現的第3型表皮細胞膜蛋白質與第2型表皮細胞生長因子受體蛋白質表現量、高組織學分級以及淋巴結轉移均具有正相關性。
基於乳癌細胞與第3型表皮細胞膜蛋白質所具有的潛在臨床價值,本發明經多方嘗試後,對於抑制乳癌細胞增生、移行或侵入等癌化生物效應,提供一種標靶治療組合物,其用於抑制乳癌細胞增生、移行或侵入,其包括具有效劑量之一第3型表皮細胞膜蛋白質靶向抑制劑。
如前所述之組合物,其中,該乳癌細胞具有基因表現上調控之第3型表皮細胞膜蛋白質、上皮細胞成長因子受體(EGFR)基因、第2型表皮細胞生長因子受體基因、第3型上皮細胞成長因子受體(HER3)基因、荷爾蒙受體基因或其二以上之組合。
如前所述之組合物,其中,該乳癌細胞具有基因表現下調控之第4型上皮細胞成長因子受體(HER4)基因。
如前所述之組合物,其中,該乳癌細胞係選自由荷爾蒙受體(HR)陽性乳腺癌細胞、第2型表皮細胞生長因子受體(HER2)陽性乳腺癌細胞及三陰性乳腺癌細胞所組成之群組。
如前所述之組合物,其中,該第3型表皮細胞膜蛋白質靶向抑制劑包括寡核苷酸或核酸酶。
如前所述之組合物,其中,該第3型表皮細胞膜蛋白質靶向抑制劑係專一靶向第3型表皮細胞膜蛋白質之信使核糖核酸(mRNA)。
如前所述之組合物,其中,該寡核苷酸係選自由、RNA干擾分子、反義核酸、DNA/RNA嵌合寡核苷酸所組成之群組,其係以互補方式結合至第3型表皮細胞膜蛋白質之編碼DNA或信使核糖核酸(mRNA)。
如前所述之組合物,其中,該寡核苷酸具有一互補序列,其以互補方式結合至該信使核糖核酸(mRNA)之第61至180鹼基之區間或第241至360鹼基之區間。
如前所述之組合物,其中,該RNA干擾分子包括小型干擾核酸(siRNA)、小髮夾式核酸(shRNA)、小分子核醣核酸(miRNA)或其組合。
如前所述之組合物,其中,該互補序列包括SEQ NO:1或SEQ NO:2。
如前所述之組合物,其中,該質體DNA片段搭載以一載體,其包括一啟動子,配置於該質體DNA片段之上游以增幅該RNA干擾分子或該反義核酸之生產。
如前所述之組合物,其中,該質體DNA片段包括SEQ NO:3或SEQ NO:4。
另一方面,為具體實現前述標靶治療組合物於臨床乳癌病患之應用,本發明進一步提供了一種標靶治療組合物於抑制乳癌細胞增生、移行或侵入之用途,其包括投予一如前所述之標靶治療組合物至一所需個體,其中,該所需個體包括具有乳癌細胞之哺乳動物。
如前所述之用途,其中,該用途係依序或分開投予該標靶治療組合物及二甲雙胍至該所需個體,其為於二甲雙胍排出該所需個體後投予該標靶治療組合物至該所需個體、或於投予二甲雙胍至該所需個體24小時後再投予該標靶治療組合物至該所需個體。
如前所述之用途,其中,該所需個體患有糖尿病。
如前所述之用途,其中,該標靶治療組合物可與一抗糖尿病藥物組合使用以投予至該所需個體,其中,該所需個體患有糖尿病。
如前所述之用途,其中,該抗糖尿病藥物係一或多種選自α-葡萄糖苷酶抑製劑(Alpha-glucosidase inhibitors)、胰島素增敏劑(Thiazolidinediones)、磺醯脲(sulphonylurea)及美格替耐(Meglitinides)所組成之群組。
如前所述之用途,其中,該標靶治療組合物可進一步與一胰島素依序、同時或分開投予該所需患者,其中,該胰島素係速效型胰島素、短效型胰島素、中效型胰島素、長效型胰島素或其二以上之組合。
如前所述之用途,其中,該所需個體為人類(Homo sapiens)。
如前所述之用途,其中,該標靶治療組合物投予以調控該乳癌細胞之上皮-間質轉化(EMT)細胞標記基因或幹細胞特性標記基因。
如前所述之用途,其中,該上皮-間質轉化(EMT)細胞標記基因包括SOX2基因、Nanog基因、EpCAM基因、Oct4基因或其二以上之組合。
如前所述之用途,其中,該幹細胞特性標記基因包括fibronectin基因、twist1/2基因、vimentin基因、E-Cadherin基因或其二以上之組合。
如前所述之用途,其中,該標靶治療組合物投予以阻斷該乳癌細胞之FAK/c-Src訊息傳遞路徑。
如前所述之用途,其中,該FAK/c-Src訊息傳遞路徑係選自由FAK-Src-RhoA、Src-JAK2-STAT2、Src-Ras/Raf-1及ERK1/2訊息傳遞路徑所組成之群組。
圖1係說明本發明所提供之標靶治療組合物於第3型表皮細胞膜蛋白質之編碼DNA或信使核糖核酸序列(mRNA)上之靶向區域;圖2A為一西方氏墨點圖,用以評估外源性第3型表皮細胞膜蛋白質於Hs578T細胞株中過量表現之情形;圖2B為一折線圖,用以說明過量表現第3型表皮細胞膜蛋白質之Hs578T細胞於離體環境中的增生能力;圖2C說明過量表現第3型表皮細胞膜蛋白質對於Hs578T細胞於離體環境中細胞遷移能力之影響;圖2D說明過量表現第3型表皮細胞膜蛋白質對於Hs578T細胞於離體環境中細胞入侵能力之影響;圖3A為一細胞傷癒實驗之影像,用以說明過量表現第3型表皮細胞膜蛋白質對於Hs578T細胞遷移能力之影響;圖3B為一長條圖,說明細胞傷癒實驗之分析結果;圖4為一西方氏墨點圖,用以說明離體人類乳癌細胞中第3型表皮細胞膜蛋白質與HER家族及荷爾蒙受體之間的串擾;圖5A為一細胞影像,係呈現過量表現第3型表皮細胞膜蛋白之HS578T細胞之細胞形態;圖5B為西方氏墨點圖,用以評估Hs578T細胞於過量表現第3型表皮細胞膜蛋白質後,第3型表皮細胞膜蛋白質、幹細胞標記以及上皮-間質細胞標記之表現量;
圖5C為西方氏墨點圖,用以評估Hs578T細胞於過量表現第3型表皮細胞膜蛋白質後,第3型表皮細胞膜蛋白質、纖網蛋白(fironectin)、Twist1/2、中間絲蛋白(vimentin)及E型鈣黏蛋白(E-cadherin)之表現量;圖6為一西方氏墨點圖,用以說明第3型表皮細胞膜蛋白質在離體環境中對於FAK/c-Src訊息調控路徑之影響;圖7A為一折線圖,呈現MTT測定之細胞增生速率;圖7B說明以Ki-67指數測定暫時性轉染後第3型表皮細胞膜蛋白質對於細胞增生的影響程度;圖8係呈現第3型表皮細胞膜蛋白質對於離體細胞遷移之影響,採用Boyden chamber assay以測定離體細胞遷移;圖9為一西方氏墨點圖,其說明第3型表皮細胞膜蛋白質於人類乳癌細胞中對於EGFR、HER2、HER3及p-Src表現量之具體調控;圖10說明第3型表皮細胞膜蛋白質對於,乳癌細胞於體內環境中致瘤性(tumorigenicity)之影響;圖11A為一免疫組織染色圖,用以評估SKBR3/Vec細胞於SCID小鼠模型中,生成腫瘤之細胞膜表面上EMP3、EGFR、HER2之表現量;圖11B為一免疫組織染色圖,用以評估SKBR3/siEMP3細胞於SCID小鼠模型中,生成腫瘤之細胞膜表面上EMP3、EGFR、HER2之表現量;圖12A為一折線圖,其用以評估SKBR3/Vec及SKBR3/siEMP3細胞在二甲雙胍處理下的半存活濃度IC50;及圖12B為一西方氏墨點圖,其說明二甲雙胍對於SKBR3細胞內AMPK訊息傳遞路徑之具體影響。
本發明之第一實施態樣在於提供一種標靶治療組合物,其用於抑制乳癌細胞增生、移行或侵入,其包括具有效劑量之一第3型表皮細胞膜蛋白質靶向抑制劑,其中,該第3型表皮細胞膜蛋白質靶向抑制劑包括但不限於寡核苷酸(oligonucleotide)或核酸酶。
作為實施例,該寡核苷酸係選自由質體DNA片段、RNA干擾分子、反義核酸、DNA/RNA嵌合寡核苷酸所組成之群組;於該些實施例中,該寡核苷酸具有一互補序列,其係以互補方式結合至第3型表皮細胞膜蛋白質之DNA或信使核糖核酸(mRNA),以達到誘導第3型表皮細胞膜蛋白質基因沉默(gene silence)或蛋白質轉譯;以RNA干擾分子(RNA interference,RNAi)為例,作為本領域公知常識,其透過雙股RNA來誘發的基因沉默(gene silence),當細胞導入了與內源性mRNA編碼區同源的雙股RNA時,所述雙股RNA具備的互補序列將與同源mRNA結合,導致mRNA降解而致使基因表現沉默。
於本實施態樣中,該RNA干擾分子包括小型干擾核酸(siRNA)、小髮夾式核酸(shRNA)、小分子核醣核酸(miRNA)或其組合,其中,該些RNA干擾分子係專一靶向第3型表皮細胞膜蛋白質之編碼DNA或信使核糖核酸(mRNA);以小型干擾核酸(siRNA)為例,其由前驅雙股RNA複合體降解而成,一般為長度23至35nt左右的小RNA分子,通過序列互補與mRNA結合,從而引起mRNA降解;除了siRNA之外,小分子RNA(microRNA)同樣也能引起RNA干擾現象,其大多為20至22nt長的核酸分子;再者,作為小分子RNA的前體,髮夾式核酸(shRNA)於第3型表皮細胞膜蛋白質的基因沉默方面具有相似的效果,其具有一莖環結構,形似於髮夾。
在一些具體的實施例中,該些寡核苷酸藉由該互補序列以互補方式結合至該信使核糖核酸(mRNA),其為編碼有第3型表皮細胞膜蛋白質之核糖核酸序列;在本實施態樣中,以人類為例,其參考序列之Genbank ID為NM_001425.3,該些寡核苷酸的靶向區域如圖1所示,分別標記為A靶向區及B靶向區,其中該A靶向區為該信使核糖核酸(mRNA)之第61至180鹼基之區間,該B靶向區該信使核糖核酸(mRNA)之第241至360鹼基之區間;在一些較佳實施例中,該互補序列包括SEQ NO:1或SEQ NO:2,分別靶向該A靶向區及該B靶向區。
於本實施方式中,除了RNA干擾分子或反異核酸等裸露核酸分子的形式投予之外,更可透過一表現載體搭載編碼有該些裸露核酸分子之編碼序列,並以轉型、轉染、轉導、基因敲入等公知生物技術將此表現載體送入所需個體之核酸轉譯系統中,以使所需個體自行產生該些RNA干擾分子或反義核酸,並達到抑制第3型表皮細胞膜蛋白質的效果;具體而言,該載體可以列舉如病毒載體、裸露的DNA或RNA表現載體、質體、黏接質體、嗜菌體載體、陽離子凝聚劑結合之DNA或RNA表現載體、包覆於脂質體中的DNA或RNA表現載體等,並不以此為限;在多個實施例中,該質體DNA片段搭載以一表現載體,其包括一啟動子,配置於該質體DNA片段之上游以增幅該RNA干擾分子或該反義核酸之生產,其中該啟動子意指啟動核酸轉錄之核酸序列,其進一步與誘導型啟動子、促進子,配置以操作下游質體DNA片段進行基因轉錄程序;在一些較佳實施例中,該質體DNA片段包括SEQ NO:3或SEQ NO:4。
另一方面,於本實施態樣中,該標靶治療組合物係針對具有下述特徵之乳癌細胞,其包括具有基因表現上調控之第3型表皮細胞膜蛋白質(EMP3)、上皮細胞成長因子受體(EGFR)基因、第2型表皮細胞生長因子受體(HER2)基因、第3型上皮細胞成長因子受體(HER3)基因、荷爾蒙受體基因(HR)、或下調控之第4型上皮細胞成長因子受體(HER4)基因;更具體地,該些乳癌細胞
係選自由荷爾蒙受體(HR)陽性乳腺癌細胞、第2型表皮細胞生長因子受體(HER2)陽性乳腺癌細胞及三陰性乳腺癌細胞所組成之群組;須說明的是,所謂三陰性乳腺癌細胞係指該些乳癌細胞的第二型人類上皮生長因子受體(HER2)、荷爾蒙受體(HR)包括動情素受體、黃體素受體於細胞中表現均呈現陰性;近期研究中更指出,除了前述三陰性乳腺癌細胞的分類以外,尚存在其他分類,本發明並不以此為限。
本發明的另一實施態樣為具體實現應用前述標靶治療組合物於臨床乳癌病患,是以進一步提供了一種標靶治療組合物於抑制乳癌細胞增生、移行或侵入之用途,其包括投予一如前所述之標靶治療組合物至一所需個體,其中,該所需個體包括具有乳癌細胞之哺乳動物。
作為實例,該用途係依序或分開投予該標靶治療組合物及二甲雙胍至該所需個體,其為於二甲雙胍排出該所需個體後投予該標靶治療組合物至該所需個體、或於投予二甲雙胍至所需個體24小時後再投予該標靶治療組合物至該所需個體;由於在所需個體上同時投予二甲雙胍進行及該標靶治療組合物進行乳癌治療方面,二甲雙胍導致所需個體中的乳癌細胞對於該標靶治療組合物的敏感度下降,從而減弱其治療效果;是以,於該用途中以依序或分開投予之方式,於所需個體身上搭配使用該標靶治療組合物及二甲雙胍。
此外,作為先前技術的一部份,已知二甲雙胍(Metformin)或其鹽類對於肌肉細胞的葡萄糖攝取與分解具有促進效果,減少了肝臟生成葡萄糖且證實有抗高血糖的效果,為現今廣泛用於治療第二型(非胰島素依賴型)糖尿病的一線藥物;在該些第二型糖尿病的患者當中,同時罹患結直腸癌、乳腺癌或胰腺癌之一的族群比例異常地高,而目前已有研究指出,二甲雙胍或其鹽類在控制第二型糖尿病患者的患癌風險具有顯著效益;然而,經本發明人驗證,本發明所提
供的標靶治療組合物在實施於所需個體身上時,需依序或分開投予以該標靶治療組合物及二甲雙胍,以減少其交互干擾所導致的脫靶效應(off-target effect)。
由於上述脫靶效應,罹患有糖尿病的乳癌患者必須依序或分開使用該標靶治療組合物及二甲雙胍,或避免使用二甲雙胍而採用其他抗糖尿病藥物或胰島素,其中抗糖尿病藥物例如一或多種選自由α-葡萄糖苷酶抑製劑(Alpha-glucosidase inhibitors)、胰島素增敏劑(Thiazolidinediones)、磺醯脲(sulphonylurea)、美格替耐(Meglitinides)及其二以上組合所組成之群組;具體來說,該α-葡萄糖苷酶抑製劑例如acarbose或miglitol,該胰島素增敏劑例如吡格列酮(Pioglitazone)或rosiglitazone,該磺醯脲可列舉如glimepiride、glyburide、chlorpropamide和glipizide,該美格替耐可列舉如repaglinide或nateglinide;胰島素例如速效型胰島素、短效型胰島素、中效型胰島素或長效型胰島素;速效型胰島素可列舉有Insulin Glulisine、Insulin Lispro、Insulin Aspart,短效型胰島素例如Regular insulin,中效型胰島素例如NPH型人體胰島素懸液,長效型胰島素可列舉如Insulin Glargine、Insulin Detemir,並無特別限制。
於本實施態樣中,該所需個體為具有乳癌細胞之哺乳動物,其可列舉有人類、大鼠、小鼠、犬、貓、兔、羊、牛、馬等哺乳動物;具體而言,該些乳癌細胞具有基因表現上調控之第3型表皮細胞膜蛋白質(EMP3)、上皮細胞成長因子受體(EGFR)基因、第2型表皮細胞生長因子受體(HER2)基因、第3型上皮細胞成長因子受體(HER3)基因、荷爾蒙受體基因(HR)、或下調控之第4型上皮細胞成長因子受體(HER4)基因;更具體地,該些乳癌細胞為荷爾蒙受體(HR)陽性乳腺癌細胞、第2型表皮細胞生長因子受體(HER2)陽性乳腺癌細胞或三陰性乳腺癌細胞所組織群組。
作為實施例,該標靶治療組合物投予以調控該乳癌細胞之上皮-間質轉化(EMT)細胞標記基因或幹細胞特性標記基因;所謂「上皮-間質轉化(EMT)
細胞標記」係指表現於癌化細胞當中的上皮-間質轉化(EMT)標記物,例如E型細胞黏附蛋白(E-cadherin)、N型細胞黏附蛋白(N-cadherin)或中間絲蛋白(vimentin);基於本領域所公知,上皮-間質轉化(EMT)的啟動標誌著癌化細胞進入轉移(metastasis),在上皮-間質轉化的過程中,上皮細胞獲得間葉細胞(mesenchymal cell)的特徵,並開始展現較高的細胞移行(migration)與侵入(invasion)能力;具體來說,上皮-間質化的特徵在於上皮細胞標記的基因減弱(knock-down)或缺失(loss),例如細胞角質素(cytokeratin)或E型細胞黏附蛋白(E-cadherin),合並間葉細胞標記,例如N型細胞黏附蛋白(N-cadherin)、中間絲蛋白(vimentin)或纖連蛋白(fibronectin)的基因上調控;上述該些細胞標記的表現變化使得該些細胞與相鄰的細胞之間粘附力下降,且細胞外基質的降解酶也將出現分泌增加的情形,進一步會導致細胞失去基細胞(basal cell)的特性,不僅重組胞內細胞骨架(cytoskeleton)結構,更促進侵入性表徵。
再者,所謂「幹細胞標記」係指表現於癌化細胞當中的幹細胞標記物,其隱含了該些癌化細胞係由正常幹細胞所轉化而來,並與該些正常幹細胞具有相同的生物功能,例如細胞增生;另一方面,該些癌化細胞亦可能由前趨細胞重新獲得幹細胞的自我更新與分化能力,例如由成熟的體細胞重新獲得幹細胞能力,並發展成腫瘤幹細胞,該些幹細胞標記基因可以列舉如SOX2基因、Nanog基因、EpCAM基因、Oct4基因,又或者乳癌幹細胞標記基因可列舉如Aldehyde Dehydrogenase 1(A1/ALDH1A1)、BMI-1、CD24、CD44、CD133、Connexin 43/GJA1、CXCR4、DLL4、EpCAM/TROP1、ErbB2/HER2、GLI-1、GLI-2、IL-1 alpha/IL-1F1、CXCR1/IL-8RA、IL-6R alpha、Integrin alpha 6/CD49f、PON1、PTEN等基因。
進一步地,該標靶治療組合物更可投予以阻斷調控該乳癌細胞之FAK/c-Src訊息傳遞路徑;於本領域所公知,FAK/c-Src在腫瘤中的表現或活性增
加使得細胞更具侵入性,而FAK/c-Src訊息傳遞路徑廣泛地受到多種受體的啟動,例如血小板衍生生長因子受體(PDGF-R)、表皮生長因子受體(EGF-R)、成纖維細胞生長因子受體(FGF-R)、第1型胰島素生長因子受體(IGF-1R)、肝細胞生長因子(HGF-R)、CSF-1R(colony stimulating factor 1 receptor)或幹細胞因子受體(SCF-R)等,也因此FAK/c-Src複合物參與在腫瘤發生(tumorigenesis)中不同的階段,並進一步影響其生長、移行或轉移等進展;在乳癌腫瘤發展中,參與其中的FAK/c-Src訊息傳遞路徑可以列舉如FAK-Src-RhoA、Src-JAK2-STAT2、Src-Ras/Raf-1或ERK1/2等訊息傳遞路徑。
為了具體說明本發明之實施方式,特於下文列舉數個實驗例,並詳細說明該些實驗例之具體實驗條件,以進一步說明本發明所產生之具體功效,惟下列實驗例僅作為列舉說明本發明之內容,並不用於限制本發明之具體態樣。
細胞培養
選用三陰性乳癌細胞株(Triple-negative breast cancer,TNBC)包括MDA-MB-231及Hs578T作為第3型表皮細胞膜蛋白質轉染實驗宿主;MDA-MB-231細胞由國立成功大學醫學檢驗生物技術學系陳百昇教授所提供;SKBR3細胞於2019年自國立成功大學臨床醫學研究所取得;具體地,MDA-MB-231細胞於37℃、5%二氧化碳之濕潤環境中,以含有10%胎牛血清(fetal bovine serum,FBS)、1%青黴素/鏈黴素之Leibovitz’s L-15培養基進行培養;Hs578T細胞於37℃、5%二氧化碳之濕潤環境中,以含有含有0.01mg/ml之胰島素、10%胎牛血清及1%之青黴素/鏈黴素之DMEM(high glucose)培養基進行培養。
DNA構築與反轉錄病毒製備
編碼有第3型表皮細胞膜蛋白質之全長cDNA選殖入pMSCV載體,其為一反轉錄病毒表現載體,係由國立成功大學分子醫學研究所蔣輯武教授所提供;搭載了第3型表皮細胞膜蛋白質之全長cDNA的pMSCV載體以磷酸鈣
(calcium phosphate)方法轉染至GP2-293細胞;轉染後培養該GP2-293細胞達48小時候,收集其培養液,並於1500rpm離心該培養液5分鐘以取得含有病毒顆粒之上清液;另一方面,具有過量表現第3型表皮細胞膜蛋白質之穩定細胞株,則以5mg/mL之嘌呤黴素(puromycin)進行選殖培養。
第3型表皮細胞膜蛋白質miRNA表現載體構築
質體pcDNA6.2-GW/EmGFP-miR-EMP3以BLOCK-iT Pol II miR RNAi Expression Vector Kits(Invitrogen)構築而得;線性化的載體pcDNA6.2-GW/EmGFP-miR與具有第3型表皮細胞膜蛋白質信使RNA之第283至532鹼基對之核苷酸序列粘合之互補序列,使其所產生之miRNA對於第3型表皮細胞膜蛋白質之編碼序列(coding sequence,CDS)具靶向性,其中第3型表皮細胞膜蛋白質信使RNA參考序列之GenBank ID為NM_001425.3;於下文實驗例中,選用的第3型表皮細胞膜蛋白質靶向miRNA序列分別標記為KDK-EMP3-1及KDK-EMP3-2,其具體序列如下表1所示,其中陰性對照組標記為Scramble,對於現存已知的任一脊椎動物基因均不具靶向性。
西方氏墨點法
以含有蛋白酶抑制劑之RIPA(radioimmunoprecipitation assay)裂解緩衝液裂解細胞,並提取蛋白質;西方氏墨點法為本領域所習知用於檢驗蛋白
質表現之技術,細節於此不詳述;簡言之,將總量30微克的蛋白質以膠體濃度6至12%的SDS-PAGE進行梯度膠體電泳分析(gradient gel electrophoresis)後,轉印至孔徑0.2至0.45微米的PVDF轉漬膜上,接著以5%無脂牛奶-TBST(Tris Buffered Saline Buffer with Tween 20)進行阻斷(blocking);轉漬膜與一級抗體在4℃雜交過夜;隔日,以TBST緩衝液清洗轉漬膜3次,轉漬膜再與二級抗體在室溫下雜交反應1小時;最後,利用化學冷光系統(Enhanced chemiluminescence,ECL)偵測蛋白質條帶。
上述一級抗體包括了第3型表皮細胞膜蛋白質(EMP3)抗體、β-肌動蛋白(β-actin)抗體、表皮生長因子受體(Epidermal growth factor receptor,EGFR)抗體、第2型表皮細胞生長因子受體(HER2)抗體、第3型表皮細胞生長因子受體(HER3)抗體、第4型上皮細胞成長因子受體(HER4)抗體、黃體素抗體(1:1000)、雌激素抗體,其稀釋比例均為1:1000;其中β-肌動蛋白抗體係作為內控制組。
MTT測試
為了進一步探索第3型表皮細胞膜蛋白(EMP3)在乳癌細胞存活率的影響,以MTT(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)測試進一步針對轉染後的細胞;首先,轉染後的細胞以100微升的培養液懸浮約1000個細胞並播種於96孔盤中的任一孔中,分別培養24、48、72及96小時;接著,以每一孔50微升之MTT試劑與細胞於37℃的暗室中反應4小時;最後,以酵素標記測量儀(enzyme-labeled meter)量測590奈米之吸光度,以評估溶解於DMSO之中的甲臢(formazan)的含量,作為乳癌細胞存活率之參考指標,以上測試共進行了四組獨立實驗。
細胞遷移與侵入測試(cell migration and invasion assay)
遷移與侵入實驗以Boyden小室(Cat # 3422,Corning Costar,Pittsburgh,PA)進行,其具有Transwell濾膜插件。簡言之,2.5×104個細胞播種於
24孔盤上的Transwell小室(孔徑8微米)中,或播種於塗佈了Matrigel之小室中以進行侵入測試(invasion assay),並以含有10%胎牛血清之完全培養液(complete medium)分別培養達48及72小時,以進行遷移與侵入測試;在Transwell小室中,未於濾膜下表面的細胞以2%之結晶紫(crystal violet)染色,而未穿過濾膜的細胞則全數洗去;最後,在光學顯微鏡下計算遷移與侵入的細胞數量,以計算每一個小室中任5個視野下細胞數量之平均數(means±SE)作為遷移與侵入的細胞數量評估依據。
反轉錄聚合酶鏈鎖反應(RT-PCR)及即時PCR(real-time PCR)分析
以RT-PCR檢驗整聯蛋白(integrin)的表現量;簡言之,2微克RNA用於反轉錄合成,以oligo dT及莫洛尼鼠白血病病毒(Moloney murine leukemia virus)反轉錄酵素(Promega Technology Enterprise Co.,Ltd.,Taipei,Taiwan)以合成第一股cDNA並儲存於-20℃;以cDNA合成套組(Promega)由總RNA合成cDNA,並以SYBR® Green Master Mix(Applied Biosystems,Foster City,CA)進行PCR擴增以檢驗整聯蛋白(integrin)之表現量;同樣地,β-肌動蛋白作為內控制組。
傷癒實驗(Wound-healing assay)
為了分析細胞於離體條件中的遷移能力,以培養插件(culture-insert)進行傷癒實驗;所述培養插件具有以500微米厚之隔板分隔之二培養孔;實驗流程簡述於此,將培養插件放置於6公分培養盤中,並輕壓其頂部以確保其緊密貼合於培養底部;種植具有過度表現第3型表皮細胞膜蛋白質之細胞株,其包括Hs578T、MDA-MB231細胞,並分別以2x105的細胞密度播種於每一培養孔中並培養過夜;接著,移除培養插件並繼續培養細胞,分別於第0、5、10及15小時,在顯微鏡(Nikon TE300,Tokyo,Japan)下記錄無細胞間隙之寬度,並以細胞覆蓋於無細胞間隙中的面積做為細胞離體遷移能力評估的參考依據,其係將每一處置的細胞在侵入無細胞間隙的程度,與0小時的時點進行比較。
體內異體移植模型(in vivo xenograft model)
以6週齡之雄性NOD/SCID小鼠進行體內異體移植模型之建構,小鼠係購自成功大學實驗動物中心,其於具有規律光週期及恆溫之無菌設施中養殖;所述實驗流程遵守中華民國動物保護法,並已獲得國立成功大學動物照護與使用委員會之批准(108196)。
將三陰性乳癌細胞株(TNBC)注射於小鼠背部兩側之皮下,每一處皮下注射以200微升之生理食鹽水懸浮1×107個TNBC細胞;自每一實驗組取4隻小鼠、8個腫瘤,並每週記錄小鼠體重與腫瘤尺寸,其中腫瘤尺寸直接以尺規測量;在皮下注射後養殖達90天,切除腫瘤後拍照並記錄其重量。
統計分析
下文實驗例中所有數據以平均值±標準差(mean±standard deviation)呈現,並以ANOVA、學生t檢定或Mann-Whitney檢定分析比較;整體存活率以對數秩測試(Log Rank tests)進行分析。
實驗例1:第3型表皮細胞膜蛋白質於離體人類乳癌之生物效應
請參閱圖2A至2D,其說明了第3型表皮細胞膜蛋白質對於乳癌細胞株於離體環境中細胞生長、細胞遷移以及細胞轉移的影響;如圖2A所示,穩定細胞株Hs578T-EMP3-1及Hs578T-EMP3-2,其相對於控制組細胞(Hs578T-Vec)展現了較高的第3型表皮細胞膜蛋白質表現量;再如圖2B所示,與控制組細胞比較,過量表現第3型表皮細胞膜蛋白質對於Hs578T細胞株之離體環境細胞增生具有劑量依賴性的促進效果(*P<0.05;**P<0.01;***P<0.001;two-way ANOVA)。
除此之外,如圖2C至2D所示,與控制組細胞相比,Hs578T-EMP3穩定細胞株在Transwell測試中,均展現了較高的細胞遷移能力(*P<0.05;雙尾t
檢定),並在Matrigel實驗中展現出更高的細胞侵入能力(**P<0.01;*P<0.05,雙尾t檢定)。
進一步地,第3型表皮細胞膜蛋白質對於細胞遷移能力的影響以傷癒實驗(Wound-healing experiment)進行測試;請參閱圖3A至3B,其係說明傷癒實驗評估第3型表皮細胞膜蛋白質對於乳癌細胞遷移能力之具體影響;如圖2A所示,Hs578T細胞以2×105cells/insert的密度培養,並以培養插入物(culture insert)製造傷口;傷口邊緣之間的面積分別在第3型表皮細胞膜蛋白質轉染後5、10、15小時計算,用以評估細胞遷移之能力;如圖3B所示,其係說明了前述傷口邊緣之間的面積計算結果,經Image J軟體分析圖3A之影像,以每一長條代表4個獨立實驗之平均數值及標準差,由圖3A至3B所示,第3型表皮細胞膜蛋白質的過量表現,相較於控制組,明顯地促進了細胞遷移(P值分別為0.00592以及0.0016),與控制組細胞相比,Hs578T-EMP3穩定細胞株關閉傷口的速度較快(p<0.01)。
於其他方面,在MDA-MB-231細胞株中也觀察到第3型表皮細胞膜蛋白質對於離體細胞生長、細胞遷移及細胞侵入具有正面效益。
實驗例2:第3型表皮細胞膜蛋白質與HER家族於人類乳癌細胞中的串擾(cross-talk)
為了驗證在離體環境中,第2型表皮細胞生長因子受體與其他次家族成員及荷爾蒙受體之間具有功能性串擾(functional cross-talk),進一步在穩定細胞株中評估第2型表皮細胞生長因子受體的表現量與其他家族成員之間表現量的相關性。
第3型表皮細胞膜蛋白質過量表現穩定細胞株Hs578T,在還原(reducing)環境中以6至12%的SDS-PAGE進行梯度分離,並以適當的抗體偵測;
如圖4所示,EMP3的過量表現對於EGFR、HER2、HER3、HER4以及荷爾蒙受體均具有劑量依賴型的上調控效果,而前述實驗結果係通過3重複實驗驗證;然而,第3型表皮細胞膜蛋白質的過量表現顯著的啟動了EGFR、HER2及HER3的基因表現,但下調節了HER4;黃體素受體的表現雖有溫和的提升,但尚未觀察到雌激素受體有任何明顯的效應;在MDA-MB-231細胞亦觀察到相似的效應;基於前述結果,第3型表皮細胞膜蛋白質表現可能參與在離體人類乳癌細胞中HER家族的反向啟動(transactivation)。
實驗例3:第3型表皮細胞膜蛋白質於乳癌細胞EMT之生物效應
請參閱圖5A至5C,其說明了第3型表皮細胞膜蛋白質對於Hs578T之上皮-間質轉化與幹細胞特性之影響;如圖5A所示,與控制組細胞比較,Hs578T-EMP3穩定細胞株展現了類纖維母細胞、細長之細胞形態,且Hs578T-EMP3-2的形態更加顯著者;因此,將進一步地檢驗第3型表皮細胞膜蛋白質對於乳癌細胞EMT的潛在生物效應;如圖5B至5C所示,第3型表皮細胞膜蛋白質的過量表現上調節了纖網蛋白(fironectin)、Twist1/2及中間絲蛋白(vimentin),但抑制了E型鈣黏蛋白(E-cadherin)的表現;除此之外,第3型表皮細胞膜蛋白質也啟動了幹細胞標記分子的表現,例如SOX2、Nanog、EpCAM及Oct4。
實驗例4:人類乳癌細胞中受到第3型表皮細胞膜蛋白質調控之訊息傳遞路徑
由於第2型表皮細胞膜蛋白質(EMP2)與整聯蛋白αV及β3之間的串擾,已被報導參與在尿路上皮細胞(urothelial cell)的離體環境細胞貼附及細胞遷移的調控之中;因此,前述生物效應將於細胞層次上,在乳癌細胞中進行調查。
於實驗例4中,Hs578T-EMP穩定細胞株3的細胞萃取物在還原條件下以6至12%的SDS-PAGE進行梯度分離,並分別偵測EGFR、EMP3、FAK、Src、phospho-Src、ROCK1、ROCK2、β-actinin、phospho-MBS、JAK2、phospho-JAK2、STAT3、RAS、RAF-1、ERK1/2、PI3K、AKT以及mTOR,其中β-actin為加載控制(loading control),而條帶之密度經量化後以相對於控制組細胞之比率呈現。
如圖6所示,以免疫墨點法呈現了第3型表皮細胞膜蛋白質對於FAK-Src-RhoA、Src-JAK2-STAT3、Src-Ras/Raf-1及ERK1/2的表現具有上調控的效應;總地來說,第3型表皮細胞膜蛋白質啟動了離體人類乳癌細胞的FAK/c-Src訊息傳遞路徑。
實驗例5:第3型表皮細胞膜蛋白質靶向對於人類乳癌細胞的離體生物效應
如前所述,第3型表皮細胞膜蛋白質作為人類乳癌細胞的致癌基因(oncogene),並可反向啟動(transactivate)HER受體基因表現,並對於多條訊息傳遞路徑具有顯著影響;為轉譯至乳癌病患照護,於實驗例6中進一步評估及確立第3型表皮細胞膜蛋白質作為標靶藥物的潛能。
於實驗例5中,選用HER3過量表現細胞株SKBR3以測試EMP3做為標靶藥物之潛能;如圖7A至7B所示,其係說明第3型表皮細胞膜蛋白質針對乳癌細胞在離體環境中細胞增生之影響;通過MTT測試,針對第3型表皮細胞膜蛋白質的基因減弱顯著的壓制了SKBR3細胞株的離體細胞增生能力(P<0.0001,two-way ANOVA),且與SKBR3/Vec控制組細胞相比,Ki-67指數同樣受到了顯著的抑制(P<0.005,paired t-test)。
進一步地,如圖8所示,其說明了上述抑制細胞增生能力對於細胞遷移的影響,與控制組相比,第3型表皮細胞膜蛋白質基因減弱相當顯著地抑制了SKBR3的細胞遷移(P<0.005,雙尾t檢定;放大倍率100X);再者,如圖9所示,EGFR、HER2、HER3及p-Src的表現量同樣在第3型表皮細胞膜蛋白質基因減弱之SKBR3穩定細胞株中受到了下調控。
實驗例6:第3型表皮細胞膜蛋白質對於人類乳房細胞靶向性之異體移植模型
執行SCID動物實驗以檢驗第3型表皮細胞膜蛋白質對於人類乳癌細胞的靶向性;如圖10所示,SKBR3/siEMP3穩定細胞株於小鼠皮下發展出尺寸較小的腫瘤(P<0.001);具體來說,選用適宜的穩定細胞株如SKBR3/V或SKBR3/siEMP3細胞,並以1x107的劑量對於SCID小鼠進行皮下注射,用以評估第3型表皮細胞膜蛋白質與第2型表皮細胞生長因子受體(HER2)協作與否的條件下之致癌潛力;經皮下注射後,每23天測量一次腫瘤尺寸及重量;如圖10所示,SKBR3/siEMP3細胞的腫瘤重量,顯著低於SKBR3/Vec細胞(**P<0.001)。
換句話說,經皮下注射的SKBR3/siEMP3細胞於小鼠背部發展出了較控制組細胞尺寸要小的腫瘤;另一方面,包括EMP3、EGFP及HER2在內的生物標記,其表現量以免疫組織染色進一步檢驗;如圖11A所示,控制組SKBR3/Vec細胞所生成的腫瘤當中,EMP3、EGFR及HER2在細胞膜上呈現了相當强表現量;然而,如圖11B所示,在SKBR3/siEMP3細胞株所生長出的腫瘤細胞中,該些生物標記分子的表現量均受到了顯著的抑制;所有的腫瘤組織接受全面性的評估並評分以呈現染色結果;具體地,透過免疫染色評估該些生物標記分子的表現量,如有不足5%之腫瘤細胞表現EGFR、HER2及HER3或缺乏任一免疫反應之腫瘤,即界定為未表現;該些有5%以上具有免疫反應者則界定為有表現。於實驗例6中,以染色之腫瘤細胞占比為基準的二級評分系統用來評估第3型表
皮細胞膜蛋白質;其中,所謂「有表現」係指涉超過20%的腫瘤細胞有第3型表皮細胞膜蛋白質免疫染色,而所謂「未表現」係指涉低於20%的腫瘤細胞有第3型表皮細胞膜蛋白質免疫染色。
實驗例7:第3型表皮細胞膜蛋白質-相關乳癌化學預防模型
二甲雙胍做為成人及小兒第二型糖尿病的一線口服抗糖尿病藥物,已知可促進肝臟細胞及胰臟細胞的減脂與提高胰島素敏感性;在離體環境(in vitro)與體內環境(in vivo)的實驗中,均顯示了二甲雙胍具有阻擋前癌症病灶(pre-cancer lesion)進展為侵入性腫瘤;因此,對於乳房癌化的預防,二甲雙胍應具有相當的潛在應用性。
然而,於實驗例7的驗證中顯示,SKBR3/Vec細胞相較於SKBR3/siEMP3細胞對於二甲雙胍的處理更加敏感,這顯示了二甲雙胍可能對於人類乳癌細胞中與第3型表皮細胞膜蛋白質相關路徑或機轉具有脫靶效應(off-target effect);請參閱圖12A至12B,其說明了第3型表皮細胞膜蛋白質對於SKBR3細胞之二甲雙胍敏感性之潛在影響;如圖12A所示,以不同濃度之二甲雙胍處理SKBR3/Vec及SKBR3/siEMP3,並於處理後72小時分別測定二甲雙胍SKBR3/Vec及SKBR3/siEMP3之細胞存活率以計算其半數抑制濃度(IC50);由圖12A可見控制組細胞之IC50較EMP3基因減弱細胞為低,顯然經EMP3基因減弱後乳癌細胞在二甲雙胍的作用存活率更高,降低了乳癌細胞對於二甲雙胍的敏感性;進一步地,如圖12B所示,二甲雙胍具體下調控了SKBR3細胞AMPK-α1及AMPK-α2的表現量,為本領域所公知者,干預AMPK及Wnt/β-catenin訊息傳遞路徑之間的相互作用,對於乳腺癌細胞生長具有其破壞性,於實驗例7中進一步地驗證二甲雙胍於SKBR3/Vec及SKBR3/siEMP3細胞中對於AMPK的
干預程度,顯示了EMP3基因減弱的乳癌細胞株在二甲雙胍的作用下,不僅細胞存活率高於控制組細胞,更在AMPK訊息傳遞路徑層面展現了對於二甲雙胍處理的低敏感性。
本發明所提供的標靶治療組合物,對於乳癌細胞具有靶向性,針對第3型表皮細胞膜蛋白質具有抑制效果,並擾亂乳癌細胞關於細胞增生、細胞移行及入侵等生物功能,可以具體減緩乳癌細胞於體內環境中的增長速度及腫瘤轉移。
本發明所提供的標靶治療組合物,可與二甲雙胍搭配治療乳癌病患,利用第3型表皮細胞膜蛋白質的靶向性,降低乳癌細胞的增生速度,依序或分開搭配二甲雙胍降低乳癌細胞之存活度,實現減緩乳癌腫瘤的增長及轉移。
本發明所提供的標靶治療組合物,除了降低乳癌細胞增生及轉移之外,更可與作為一線抗糖尿病藥物之二甲雙胍,搭配使用於患有糖尿病之乳癌患者,達到糖尿病控制及乳癌治療,提供乳癌患者更多醫療選項。
TW202310850A_111129484_SEQL.xml
Claims (8)
- 一種標靶治療組合物,其用於抑制乳癌細胞增生、移行或侵入,其包括具有效劑量之一第3型表皮細胞膜蛋白質靶向抑制劑,其中,該第3型表皮細胞膜蛋白質靶向抑制劑包括寡核苷酸,其是以互補方式結合至該信使核糖核酸(mRNA),其中,該寡核苷酸是由如SEQ ID NO:1或SEQ ID NO:2之鹼基序列所組成。
- 如請求項1所述之組合物,其中,該乳癌細胞係選自由荷爾蒙受體(HR)陽性乳腺癌細胞、第2型表皮細胞生長因子受體(HER2)陽性乳腺癌細胞及三陰性乳腺癌細胞所組成之群組。
- 如請求項1所述之組合物,其中,該寡核苷酸係選自由RNA干擾分子及反義核酸所組成之群組。
- 如請求項1所述之組合物,其中,該寡核苷酸係以互補方式結合至該信使核糖核酸(mRNA)之第61至180鹼基之區間或第241至360鹼基之區間。
- 如請求項1所述之組合物,其中,該寡核苷酸之生產係增幅自一質體DNA片段,其中,該質體DNA片段是由SEQ ID NO:3或SEQ ID NO:4之鹼基序列所組成,其中,該質體DNA片段搭載以一載體,其包括一啟動子,配置於該質體DNA片段之上游以增幅該寡核苷酸之生產。
- 一種標靶治療組合物用於製備抑制乳癌細胞增生、移行或侵入之醫藥組合物之用途,其中,該醫藥組合物包括一如請求項1所述之標靶治療組合物,該用途包括投予該醫藥組合物至一所需個體,其中,該所需個體包括具有乳癌細胞之哺乳動物。
- 如請求項6所述之用途,其中,該醫藥組合物之投予係依序或分開投予該標靶治療組合物及二甲雙胍至該所需個體,其為於二甲雙胍排出該所 需個體後投予該標靶治療組合物至該所需個體、或於投予二甲雙胍至該所需個體24小時後再投予該標靶治療組合物至該所需個體。
- 如請求項7所述之用途,其中,該所需個體患有糖尿病。
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