Murata Mayu, Kuwajima Hiroki, Tanaka Junna, Hasegawa Nanami, Yuki Ryuzaburo, Saito Youhei, Nakayama Yuji
Laboratory of Biochemistry and Molecular Biology, Kyoto Pharmaceutical University, Kyoto, Japan.
Cell Biochem Funct. 2024 Dec;42(8):e70022. doi: 10.1002/cbf.70022.
TNK2 is a ubiquitously expressed nonreceptor-type tyrosine kinase. TNK2 participates in tumorigenesis, and TNK2 activation has been found in various cancers; therefore, TNK2 is a promising target for cancer chemotherapy. While the TNK2 inhibitor XMD16-5 is highly selective, it inhibits cytokinesis at higher concentrations by targeting Aurora B kinase, a key enzyme for cell division. Cytokinesis failure frequently generates polyploid cells, and the surviving polyploid cells risk leading to cancer development and malignant progression via chromosome instability. In this study, to investigate the possibility that (R)-9bMS, a TNK2 inhibitor structurally related to XMD16-5, drives malignant progression by inducing abnormal cell division, we examined its effects on cell division, Aurora B autophosphorylation, and colony formation. Cell count results showed a reduction in the number of A431, HeLa S3, HCT116, and MCF7 cells upon TNK2 inhibitor treatment. Microscopic observation indicated the formation of multinucleated and nucleus-enlarged cells. An increase in DNA content was confirmed with flow cytometry, which was underpinned by an increased number of centrosomes. Time-lapse imaging revealed mitotic failure, such as mitotic slippage and cytokinesis failure, as a cause of polyploidization. Of note, TNK2 knockdown significantly increased multinucleated cells, but the effect was quite weak, suggesting that TNK2 inhibition may only partially contribute to mitotic failure and polyploidization. Expectedly, Aurora B phosphorylation was reduced by (R)-9bMS like XMD16-5, but not by TNK2 knockdown. Collectively, TNK2 inhibitors (R)-9bMS and XMD16-5 induce polyploidization via mitotic failure caused by the inhibition of Aurora B kinase rather than TNK2. Notably, (R)-9bMS treatment promoted anchorage-independent colony formation, a hallmark of cancer. Our findings suggest that (R)-9bMS at a high concentration risks promoting cancer development or malignant progression. Therefore, caution should be used when using TNK2 inhibitors for cancers where TNK2 activation is not the transforming mutation and higher concentrations of TNK2 inhibitors are required to slow proliferation.
TNK2是一种在全身广泛表达的非受体型酪氨酸激酶。TNK2参与肿瘤发生，且在多种癌症中均发现有TNK2激活现象；因此，TNK2是癌症化疗的一个有前景的靶点。虽然TNK2抑制剂XMD16 - 5具有高度选择性，但在较高浓度时，它会通过靶向细胞分裂的关键酶极光激酶B（Aurora B kinase）来抑制胞质分裂。胞质分裂失败常常会产生多倍体细胞，而存活下来的多倍体细胞有通过染色体不稳定导致癌症发展和恶性进展的风险。在本研究中，为了探究与XMD16 - 5结构相关的TNK2抑制剂（R）- 9bMS是否通过诱导异常细胞分裂来推动恶性进展，我们检测了其对细胞分裂、极光激酶B自身磷酸化以及集落形成的影响。细胞计数结果显示，用TNK2抑制剂处理后，A431、HeLa S3、HCT116和MCF7细胞的数量减少。显微镜观察表明形成了多核细胞和细胞核增大的细胞。通过流式细胞术证实了DNA含量增加，这是由中心体数量增加所支撑的。延时成像揭示有丝分裂失败，如有丝分裂滑脱和胞质分裂失败，是多倍体化的原因。值得注意的是，敲低TNK2显著增加了多核细胞，但效果相当微弱，这表明抑制TNK2可能仅部分导致有丝分裂失败和多倍体化。正如预期的那样，（R）- 9bMS像XMD16 - 5一样降低了极光激酶B的磷酸化，但敲低TNK2则没有这种效果。总体而言，TNK2抑制剂（R）- 9bMS和XMD16 - 5是通过抑制极光激酶B导致有丝分裂失败进而诱导多倍体化，而非通过抑制TNK2。值得注意的是，（R）- 9bMS处理促进了不依赖贴壁的集落形成，这是癌症的一个标志。我们的研究结果表明，高浓度的（R）- 9bMS有促进癌症发展或恶性进展的风险。因此，在使用TNK2抑制剂治疗TNK2激活不是转化突变且需要更高浓度的TNK2抑制剂来减缓增殖的癌症时应谨慎使用。
