Osipo C, Patel P, Rizzo P, Clementz A G, Hao L, Golde T E, Miele L
Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL 60153, USA.
Oncogene. 2008 Aug 28;27(37):5019-32. doi: 10.1038/onc.2008.149. Epub 2008 May 12.
ErbB-2 overexpression in breast tumors is associated with poor survival. Expression of Notch-1 and its ligand, Jagged-1, is associated with the poorest survival, including ErbB-2-positive tumors. Trastuzumab plus chemotherapy is the standard of care for ErbB-2-positive breast cancer. A proportion of tumors are initially resistant to trastuzumab and acquired resistance to trastuzumab occurs in metastatic breast cancer and is associated with poor prognosis. Thus, we investigated whether Notch-1 contributes to trastuzumab resistance. ErbB-2-positive cells have low Notch transcriptional activity compared to non-overexpressing cells. Trastuzumab or a dual epidermal growth factor receptor (EGFR)/ErbB-2 tyrosine kinase inhibitor (TKI) increased Notch activity by 2- to 6-fold in SKBr3, BT474 and MCF-7/HER2-18 cells. The increase in activity was abrogated by a Notch inhibitor, gamma-secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1trade mark precursor, increased amount and nuclear accumulation of active Notch-1(IC) and increased expression of targets, Hey1 and Deltex1 mRNAs, and Hes5, Hey1, Hes1 proteins. Importantly, trastuzumab-resistant BT474 cells treated with trastuzumab for 6 months expressed twofold higher Notch-1, twofold higher Hey1, ninefold higher Deltex1 mRNAs and threefold higher Notch-1 and Hes5 proteins, compared to trastuzumab-sensitive BT474 cells. The increase in Hey1 and Deltex1 mRNAs in resistant cells was abrogated by a Notch-1 siRNA. Cell proliferation was inhibited more effectively by trastuzumab or TKI plus a GSI than either agent alone. Decreased Notch-1 by siRNA increased efficacy of trastuzumab in BT474 sensitive cells and restored sensitivity in resistant cells. Trastuzumab plus a GSI increased apoptosis in sensitive cells by 20-30%. A GSI alone was sufficient to increase apoptosis in trastuzumab-resistant BT474 cells by 20%, which increased to 30% with trastuzumab. Notch-1 siRNA alone decreased cell growth by 30% in sensitive and more than 50% in resistant BT474 cells. Furthermore, growth of both trastuzumab sensitive and resistant cells was completely inhibited by combining trastuzumab plus Notch-1 siRNA. More importantly, Notch-1 siRNA or a GSI resensitized trastuzumab-resistant BT474 cells to trastuzumab. These results demonstrate that ErbB-2 overexpression suppresses Notch-1 activity, which can be reversed by trastuzumab or TKI. These results suggest that Notch-1 might play a novel role in resistance to trastuzumab, which could be prevented or reversed by inhibiting Notch-1.
乳腺肿瘤中ErbB-2过表达与生存率低相关。Notch-1及其配体Jagged-1的表达与最差的生存率相关，包括ErbB-2阳性肿瘤。曲妥珠单抗联合化疗是ErbB-2阳性乳腺癌的标准治疗方案。一部分肿瘤最初对曲妥珠单抗耐药，转移性乳腺癌会出现对曲妥珠单抗的获得性耐药，且与预后不良相关。因此，我们研究了Notch-1是否促成曲妥珠单抗耐药。与未过表达的细胞相比，ErbB-2阳性细胞具有较低的Notch转录活性。曲妥珠单抗或双表皮生长因子受体（EGFR）/ErbB-2酪氨酸激酶抑制剂（TKI）可使SKBr3、BT474和MCF-7/HER2-18细胞中的Notch活性增加2至6倍。Notch抑制剂γ-分泌酶抑制剂（GSI）或Notch-1小干扰RNA（siRNA）可消除活性的增加。曲妥珠单抗降低了Notch-1前体，增加了活性Notch-1（IC）的量和核积累，并增加了靶标Hey1和Deltex1 mRNA以及Hes5、Hey1、Hes1蛋白的表达。重要的是，与对曲妥珠单抗敏感的BT474细胞相比，用曲妥珠单抗处理6个月的对曲妥珠单抗耐药的BT474细胞中Notch-1高两倍、Hey1高两倍、Deltex1 mRNA高九倍、Notch-1和Hes5蛋白高三倍。Notch-1 siRNA可消除耐药细胞中Hey1和Deltex1 mRNA的增加。曲妥珠单抗或TKI加GSI比单独使用任何一种药物更有效地抑制细胞增殖。通过siRNA降低Notch-1可增加曲妥珠单抗在BT474敏感细胞中的疗效，并恢复耐药细胞的敏感性。曲妥珠单抗加GSI可使敏感细胞中的凋亡增加20%-30%。单独使用GSI足以使对曲妥珠单抗耐药的BT474细胞中的凋亡增加20%，与曲妥珠单抗联合使用时增加到30%。单独的Notch-1 siRNA可使敏感的BT474细胞中的细胞生长降低30%，使耐药的BT474细胞中的细胞生长降低超过50%。此外，曲妥珠单抗联合Notch-1 siRNA可完全抑制曲妥珠单抗敏感和耐药细胞的生长。更重要的是，Notch-1 siRNA或GSI可使对曲妥珠单抗耐药的BT474细胞对曲妥珠单抗重新敏感。这些结果表明，ErbB-2过表达会抑制Notch-1活性，而曲妥珠单抗或TKI可使其逆转。这些结果表明，Notch-1可能在对曲妥珠单抗的耐药中发挥新作用，通过抑制Notch-1可预防或逆转这种耐药。
