Manupati Kanakaraju, Hao Mingang, Haas Michael, Yeo Syn Kok, Guan Jun-Lin
Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267.
Res Sq. 2024 Apr 1:rs.3.rs-3953289. doi: 10.21203/rs.3.rs-3953289/v1.
Nuclear mitotic apparatus protein 1 (NuMA1) is a cell cycle protein and upregulated in breast cancer. However, the role of NuMA1 in TNBC and its regulation in heterogenous populations remains elusive.
We performed CRISPR mediated deletion of NuMA1 in mouse TNBC cells, BF3M. FACS was utilized to isolate BCSCs, and bulk cells based on CD29 and CD61 markers. Cell viability, migration, and invasion ability of BCSCs and bulk cells was evaluated using MTT, wound healing and transwell invasion assays, respectively. In vivo mouse breast cancer and lung metastatic models were generated to evaluate the combination treatment of SMI-4a and Lys-o5 inhibitors.
We identified that high expression of NuMA1 associated with poor survival of breast cancer patients. Further, human tissue microarray results depicted high expression of NuMA1 in TNBC relative to non-adjacent normal tissues. Therefore, we performed CRISPR mediated deletion of NuMA1 in a mouse mammary tumor cell line, BF3M and revealed that NuMA1 deletion reduced mammary tumorigenesis. We also showed that NuMA1 deletion reduced ALDH and CD29CD61 breast cancer stem cells (BCSCs), indicating a role of NuMA1 in BCSCs. Further, sorted and characterized BCSCs from BF3M depicted reduced metastasis with NuMA1 KO cells. Moreover, we found that PIM1, an upstream kinase of NuMA1 plays a preferential role in maintenance of BCSCs associated phenotypes, but not in bulk cells. In contrast, PIM1 kinase inhibition in bulk cells depicted increased autophagy (FIP200). Therefore, we applied a combination treatment strategy of PIM1 and autophagy inhibition using SMI-4a and Lys05 respectively, showed higher efficacy against cell viability of both these populations and further reduced breast tumor formation and metastasis. Together, our study demonstrated NuMA1 as a potential therapeutic target and combination treatment using inhibitors for an upstream kinase PIM1 and autophagy inhibitors could be a potentially new therapeutic approach for TNBC.
Our study demonstrated that combination treatment of PIM1 inhibitor and autophagy inhibitor depicted reduced mammary tumorigenesis and metastasis by targeting NuMA1 in BCSCs and bulk cells of TNBC, demonstrating this combination treatment approach could be a potentially effective therapy for TNBC patients.
核有丝分裂器蛋白1（NuMA1）是一种细胞周期蛋白，在乳腺癌中上调。然而，NuMA1在三阴性乳腺癌（TNBC）中的作用及其在异质性群体中的调控机制仍不清楚。
我们在小鼠TNBC细胞BF3M中进行了CRISPR介导的NuMA1基因敲除。利用荧光激活细胞分选术（FACS）基于CD29和CD61标志物分离乳腺癌干细胞（BCSCs）和普通细胞。分别使用MTT、伤口愈合和Transwell侵袭实验评估BCSCs和普通细胞的细胞活力、迁移和侵袭能力。建立体内小鼠乳腺癌和肺转移模型，以评估SMI-4a和Lys-o5抑制剂的联合治疗效果。
我们发现NuMA1高表达与乳腺癌患者的不良生存相关。此外，人体组织芯片结果显示，与非相邻正常组织相比，TNBC中NuMA1高表达。因此，我们在小鼠乳腺肿瘤细胞系BF3M中进行了CRISPR介导的NuMA1基因敲除，发现NuMA1缺失可降低乳腺肿瘤发生。我们还表明，NuMA1缺失可减少醛脱氢酶（ALDH）和CD29CD61乳腺癌干细胞（BCSCs），表明NuMA1在BCSCs中发挥作用。此外，从BF3M中分选并鉴定的BCSCs显示，NuMA1基因敲除细胞的转移减少。此外，我们发现NuMA1的上游激酶PIM1在维持BCSCs相关表型方面起优先作用，但在普通细胞中不起作用。相反，普通细胞中PIM1激酶抑制显示自噬增加（FIP200）。因此，我们分别应用SMI-4a和Lys05对PIM1和自噬进行联合抑制治疗策略，对这两种细胞群体的细胞活力显示出更高的疗效，并进一步减少乳腺肿瘤形成和转移。总之，我们的研究表明NuMA1是一个潜在的治疗靶点，使用上游激酶PIM1抑制剂和自噬抑制剂的联合治疗可能是TNBC一种潜在的新治疗方法。
我们的研究表明，PIM1抑制剂和自噬抑制剂联合治疗通过靶向TNBC的BCSCs和普通细胞中的NuMA1，减少了乳腺肿瘤发生和转移，表明这种联合治疗方法可能是TNBC患者一种潜在有效的治疗方法。
