Colunga A, Bollino D, Schech A, Aurelian L
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA.
Gene Ther. 2014 Apr;21(4):371-8. doi: 10.1038/gt.2014.6. Epub 2014 Feb 20.
Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ΔPK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0.1 pfu/cell) without resistance development and it inhibits the 3D growth potential (spheroids and agarose colonies) of melanoma and breast cancer cells. ΔPK induces calpain activation in both melanoma and breast cancer 3D cultures as determined by the loss of the p28 regulatory subunit, and 3D growth is restored by treatment with the calpain inhibitor PD150606. In melanoma, ΔPK infection also induces light chain 3 (LC3)-II accumulation and p62/SQSTM1 clearance, both markers of autophagy, and 3D growth is restored by treatment with the autophagy inhibitor chloroquine (CQ). However, expression of the autophagy-required protein Atg5 is not altered and CQ does not restore p62/SQSTM1 expression, suggesting that the CQ effect may be autophagy-independent. PD150606 restores expression of p62/SQSTM1 in ΔPK-infected melanoma cultures, suggesting that calpain activation induces anti-tumor activity through p62/SQSTM1 clearance.
溶瘤病毒疗法是一种很有前景的策略，可通过在快速增殖细胞中进行选择性病毒复制来减轻肿瘤负担。然而，维持肿瘤克隆性的缓慢复制的癌症干细胞（CSCs）的裂解相对较弱，而且程序性细胞死亡途径对溶瘤活性的潜在贡献仍知之甚少。我们发现，溶瘤病毒ΔPK能以低滴度（0.1 pfu/细胞）裂解富含CSC的乳腺癌和黑色素瘤3D球体培养物，且不会产生抗性，并且它能抑制黑色素瘤和乳腺癌细胞的3D生长潜力（球体和琼脂糖集落）。通过p28调节亚基的缺失确定，ΔPK在黑色素瘤和乳腺癌3D培养物中均诱导钙蛋白酶激活，用钙蛋白酶抑制剂PD150606处理可恢复3D生长。在黑色素瘤中，ΔPK感染还诱导自噬的两个标志物轻链3（LC3）-II积累和p62/SQSTM1清除，用自噬抑制剂氯喹（CQ）处理可恢复3D生长。然而，自噬所需蛋白Atg5的表达未改变，且CQ不能恢复p62/SQSTM1表达，这表明CQ的作用可能与自噬无关。PD150606可恢复ΔPK感染的黑色素瘤培养物中p62/SQSTM1的表达，这表明钙蛋白酶激活通过p62/SQSTM1清除诱导抗肿瘤活性。
