Al-Harbi Sayer, Choudhary Gaurav S, Ebron Jey Sabith, Hill Brian T, Vivekanathan Nagarajavel, Ting Angela H, Radivoyevitch Tomas, Smith Mitchell R, Shukla Girish C, Almasan Alex
Departments of Cancer Biology, Cleveland, OH, 44195, USA.
Department of Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Cancer, Riyadh, 11211, Saudi Arabia.
Mol Cancer. 2015 Nov 4;14:185. doi: 10.1186/s12943-015-0460-8.
BCL-xL is an anti-apoptotic BCL-2 family protein that inhibits apoptosis and is overexpressed in many cancers. We have reported that acquired resistance to the BCL-2 inhibitor ABT-199 (venetoclax) is associated with increased BCL-xL expression. Yet, how BCL-xL mediates chemoresistance in hematopoietic malignancies is not clear. This finding may help in design of new strategies for therapeutic intervention to overcome acquired chemoresistance mediated by BCL-xL.
We now show that the increased BCL-xL expression was inversely correlated with that of miR-377 in ABT-199-resistant cells. This finding was also extended to a panel of B-cell lymphoid lines and primary chronic lymphocytic leukemia (CLL) cells. miR-377 suppressed BCL-xL expression by recognizing two binding sites in the BCL-xL 3'-UTR. Mutation of these two miR-377 consensus-binding sites completely abolished its regulatory effect. Expression of a miR-377 mimic downregulated BCL-xL protein expression and significantly increased apoptotic cell death. Expression of a miR-377 inhibitor restored BCL-xL protein expression and limited cell death caused by the hypomethylating agent 5-azacytidine. Thus, miR-377-dependent BCL-xL regulation drives acquired therapeutic resistance to ABT-199. We further show that CLL patients who received a diverse array of chemotherapy regimens also had significantly higher BCL-xL and lower miR377 expression, indicating that exposure to chemotherapy might trigger transcriptional silencing of miR-377, which results in high levels of BCL-xL. Importantly, CLL patients with high BCL-xL/low miR-377 expression had an advanced tumor stage. Moreover, the high BCL-xL expression correlated with short treatment-free survival in 76 CLL patients. miR-377 is located at 14q32 in the DLK1-DIO3 region, which encodes the largest tumor suppressor miRNA cluster in humans. Examination of five additional 14q32 miRNAs revealed that the majority were significantly down-regulated in most CLL patients as well as in ABT-199-resistant cell lines. Remarkably, four of these miRNAs had significantly decreased expression in chemotherapy-treated CLL patients as compared to those untreated. These findings indicate a reduced expression of multiple miRNAs that may reflect a global silencing of this miRNA cluster in therapy-resistant lymphoid cells.
These findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies.
BCL-xL是一种抗凋亡的BCL-2家族蛋白，可抑制细胞凋亡，在许多癌症中过表达。我们曾报道，对BCL-2抑制剂ABT-199（维奈托克）产生的获得性耐药与BCL-xL表达增加有关。然而，BCL-xL如何介导造血系统恶性肿瘤的化疗耐药尚不清楚。这一发现可能有助于设计新的治疗干预策略，以克服由BCL-xL介导的获得性化疗耐药。
我们现在发现，在对ABT-199耐药的细胞中，BCL-xL表达的增加与miR-377的表达呈负相关。这一发现也扩展到一组B细胞淋巴瘤系和原发性慢性淋巴细胞白血病（CLL）细胞。miR-377通过识别BCL-xL 3'-UTR中的两个结合位点来抑制BCL-xL的表达。这两个miR-377共有结合位点的突变完全消除了其调节作用。miR-377模拟物的表达下调了BCL-xL蛋白表达，并显著增加了凋亡细胞死亡。miR-377抑制剂的表达恢复了BCL-xL蛋白表达，并限制了由低甲基化剂5-氮杂胞苷引起的细胞死亡。因此，miR-377依赖的BCL-xL调节驱动了对ABT-199的获得性治疗耐药。我们进一步表明，接受多种化疗方案的CLL患者也有显著更高的BCL-xL表达和更低的miR377表达，这表明接触化疗可能会触发miR-377的转录沉默，从而导致BCL-xL水平升高。重要的是，BCL-xL高表达/miR-377低表达的CLL患者肿瘤分期较晚。此外，在76例CLL患者中，高BCL-xL表达与无治疗生存期短相关。miR-377位于DLK1-DIO3区域的14q32，该区域编码人类最大的肿瘤抑制性miRNA簇。对另外5个14q32 miRNA的检测显示，在大多数CLL患者以及对ABT-199耐药的细胞系中，大多数miRNA显著下调。值得注意的是，与未治疗的患者相比，在接受化疗的CLL患者中，其中4种miRNA的表达显著降低。这些发现表明多种miRNA表达降低，这可能反映了该miRNA簇在治疗耐药的淋巴细胞中的整体沉默。
这些发现揭示了一种新机制，即miR-377的下调增加了BCL-xL表达，促进了B细胞淋巴瘤的化疗耐药。
