Manouchehri Jasmine M, Marcho Lynn, Cherian Mathew A
bioRxiv. 2023 Sep 17:2023.09.15.557965. doi: 10.1101/2023.09.15.557965.
Breast cancer is the leading cause of cancer-related death among women worldwide. Patients diagnosed with triple-negative breast cancer (TNBC) have limited therapeutic options that produce durable responses. Hence, a diagnosis of TNBC is associated with a poor prognosis compared to other types of breast cancer. As a result, there is a critical need for novel therapies that can deepen and prolong responses.We previously found that chemotherapy causes the release of extracellular adenosine triphosphate (eATP). Augmenting eATP release can boost the response of TNBC cells to chemotherapy and cause increased cell death. However, eATP concentrations are limited by several families of extracellular ATPases, which complicates the design of compounds that attenuate eATP degradation.In this study, we hypothesized that heparan sulfate (HS) would inhibit extracellular ATPases and accentuate chemotherapy-induced cytotoxicity in TNBC by augmenting eATP. HS can be desulfated by sulfatase 1 and 2; sulfatase 2 is consistently highly expressed in a variety of cancers including breast cancer, whereas sulfatase 1 is not. We hypothesized that the sulfatase 2 inhibitor OKN-007 would exacerbate chemotherapy-induced eATP release and TNBC cell death.
TNBC cell lines and nontumorigenic immortal mammary epithelial cells were treated with paclitaxel in the presence of heparan sodium sulfate and/or OKN-007; eATP content and cell viability were evaluated. In addition, protein and cell surface expression of sulfatases 1 and 2 were determined in all examined cell lines via ELISA, Western blot, and flow cytometry analyses.
Sulfatase 2 was highly expressed in TNBC cell lines and human breast cancer samples but not in immortal mammary epithelial cells and much less so in normal human breast tissue and ductal carcinoma in situ samples. OKN-007 exacerbated chemotherapy-induced eATP release and chemotherapy-induced TNBC cell death. When combined with chemotherapy, OKN-007 attenuated cells with a cancer-initiating cell phenotype.
These results suggest that sulfatase 2 inhibitors in combination with chemotherapy attenuate the viability of TNBC cells more than chemotherapy alone by exacerbating eATP release. These effects, as well as their capacity to attenuate the cancer-initiating cell fraction, may translate into combination therapies for TNBC that induce deeper and more durable responses.
乳腺癌是全球女性癌症相关死亡的主要原因。被诊断为三阴性乳腺癌（TNBC）的患者治疗选择有限，且难以产生持久疗效。因此，与其他类型的乳腺癌相比，TNBC的诊断意味着预后较差。所以，迫切需要能够增强和延长疗效的新型疗法。我们之前发现化疗会导致细胞外三磷酸腺苷（eATP）的释放。增加eATP的释放可以增强TNBC细胞对化疗的反应并导致细胞死亡增加。然而，eATP的浓度受到几个细胞外ATP酶家族的限制，这使得设计减弱eATP降解的化合物变得复杂。在本研究中，我们假设硫酸乙酰肝素（HS）会抑制细胞外ATP酶，并通过增加eATP来增强化疗诱导的TNBC细胞毒性。HS可被硫酸酯酶1和2去硫酸化；硫酸酯酶2在包括乳腺癌在内的多种癌症中持续高表达，而硫酸酯酶1则不然。我们假设硫酸酯酶2抑制剂OKN-007会加剧化疗诱导的eATP释放和TNBC细胞死亡。
在存在硫酸乙酰肝素钠和/或OKN-007的情况下，用紫杉醇处理TNBC细胞系和非致瘤性永生乳腺上皮细胞；评估eATP含量和细胞活力。此外，通过ELISA、蛋白质印迹和流式细胞术分析在所有检测的细胞系中测定硫酸酯酶1和2的蛋白质和细胞表面表达。
硫酸酯酶2在TNBC细胞系和人乳腺癌样本中高表达，但在永生乳腺上皮细胞中不表达，在正常人类乳腺组织和原位导管癌样本中表达更低。OKN-007加剧了化疗诱导的eATP释放和化疗诱导的TNBC细胞死亡。与化疗联合使用时，OKN-007使具有癌症起始细胞表型的细胞减少。
这些结果表明，硫酸酯酶2抑制剂与化疗联合使用比单独化疗更能通过加剧eATP释放来降低TNBC细胞的活力。这些作用以及它们减弱癌症起始细胞比例的能力，可能转化为诱导更深和更持久反应的TNBC联合治疗方法。
