利用針對HER2/neu 原致癌基因的siRNA抑制HER2/neu大量表現癌細胞的致癌能力

Abstract

HER2/neu proto-oncogene是epidermal growth factor receptor(EGFR)家族成員之一。在許多癌症如卵巢癌、乳癌、肺癌、腎癌、結腸癌、胃癌等等，常有HER2/neu大量表現的現象。HER2/neu大量表現的癌症常伴隨著較強的轉移能力、血管新生能力、抗藥性及較差治癒率等特性，因此HER2/neu成為癌症治療上一個重要的標的。目前已有許多抑制HER2/neu表現及活性的方法，經由抑制HER2/neu，這些方法皆能有效抑制HER2/neu-overexpressing癌症。siRNA是抑制基因表現的有力工具，我們實驗室已篩選到可以抑制HER2/neu的siRNA。為了提高攜帶siRNA進入細胞的效率，本實驗室利用重組腺病毒作為攜帶針對HER2/neu的 siRNA的載體(rAdv-siHER2)，並想探討rAdv-siHER2是否可作為HER2/neu-overexpressing癌症的基因療法。結果發現rAdv-siHER2可以抑制HER2/neu-overexpressing癌細胞株BT-474、MDA-MB-453、SK-OV-3-sub1及SK-OK-3-ip1中HER2/neu的表現量。rAdv-siHER2感染HER2/neu-overexpressing癌細胞株後，發現細胞週期中sub-G1比例增加及PARP出現被切割的小片段，但是在HER2/neu low-expressing癌細胞株MDA-MB-231、MCF-7、HeLa及NPC-TW04及正常細胞株ECV304並沒有這些現象。代表rAdv-siHER2可專一性地促進HER2/neu-overexpressing癌細胞走向凋亡。此外，rAdv-siHER2能有效抑制HER2/neu-overexpressing癌細胞在柔軟瓊脂中形成的聚落數，而對HER2/neu low-expressing癌細胞影響不大。rAdv-siHER2感染BT-474後，也能減少HER2/neu下游訊息傳遞分子ERK1/2及Akt被活化的現象。此外，將rAdv-siHER2注射入腫瘤中，可以抑制SK-OV-3-ip1腫瘤在NOD/SCID小鼠皮下的生長。最後，rAdv-siHER2加上抗癌藥物CDDP (cisplatin)複合處理後，更能促進 CDDP誘發HER2/neu-overexpressing癌細胞凋亡的能力。綜合以上結果， rAdv-siHER2未來有潛力開發作為治療HER2/neu-overexpressing癌細胞的基因療法之一。

HER2/neu proto-oncogene is a member of epidermal growth factor receptor family. Overexpression and amplification of HER2/neu is found in many types of human cancers, including ovary, breast, lung, kindey, colon and gastrointestinal cancers. Overexpression of HER2/neu in cancers is correlated with enhanced metastasis, angiogenesis, and chemo-resistance, and poor prognosis, therefore treatment of HER2/neu-overexpressing cancers has become an urgent issue in cancer therapy. Inhibition of HER2/neu expression has been shown to be able to suppress HER2/neu-overexpressing cancers. Many strategies have been developed to inhibit HER2/neu in hope to cure this devastating disease. In this study, we chose to use siRNA against HER2/neu to suppress HER2/neu-overexpressing cancer cells. We have constructed a recombinant adenovirus expressing HER2/neu siRNA (named rAdv-siHER2). We demonstrated that rAdv-siHER2 could effectively inhibit HER2/neu expression in various HER2/neu-overexpressing cancer cell lines. This inhibition of HER2/neu expression by rAdv-siHER2 can lead to inhibition of ERK and AKT activities, both of which are downstream signaling molecules of HER2/neu and are required for cell proliferation and survival. In accordance with its ability to inhibit HER2/neu expression, rAdv-siHER2 was shown, by sub-G1 analysis and PARP cleavage assay, to be able to specifically induce apoptosis in HER2/neu-overexpressing cancer cell lines (BT-474, MDA-MB-453, SK-OV-3-sub1 and SK-OV-3-ip1), but not in HER2/neu low-expressing cancer cell lines (MDA-MB-231, MCF-7, HeLa and NPC-TW04) and non-transformed cell line (ECV304). We also found that rAdv-siHER2 could specifically inhibit the soft-agar colony-forming ability of the HER2/neu-overexpressing cancer cell lines, but not that of the HER2/neu low-expressing cancer cell lines. Moreover, intratumoral injection of rAdv-siHER2 could effciently suppress the growth of established subcutaneous SK-OV-3-ip1 tumor on NOD/SCID mice. Finally, we demonstrated that rAdv-siHER2 could sensitize HER2/neu-overexpressing cancer cell lines, but not HER2/neu low-expressing cancer cell lines, to chemotherapeutic agent, cisplatin (CDDP). Taken together, these data suggest that rAdv-siHER2 has the potential to be developed into a gene therapy agent specifically against HER2/neu-overexpressing cancers.