■作为保乳治疗(BCT)的一部分,术后放疗是提高乳腺癌临床疗效的主要手段之一。然而,电离辐射(IR)可能会诱导BCa细胞产生辐射抗性,治疗后引起肿瘤复发和转移。最近,据报道,DOC-2/DAB2相互作用蛋白(DAB2IP)在多种癌症中经常下调,并且与肿瘤对放疗的耐受性有关。在这项研究中,引入BCa细胞系,以研究DAB2IP缺陷如何在体外和体内影响BCa细胞的放射敏感性,并讨论可能的机制。
■小RNA干扰系统(siRNA)用于降低两种BCa细胞系中DAB2IP的表达,MDA-MB-231和4T1。通过克隆形成试验或MTT方法检测对IR或抗肿瘤反应的细胞,分别。对于体内研究,将siDAB2IP或siControl细胞皮下注射到每只雌性小鼠的右侧腹中。球体形成测定,实施软琼脂集落锚定试验和体内肿瘤发生试验以检查BCa细胞的干细胞样特征。分别应用管形成测定法和免疫荧光测定法(IFA)来确定体外和体内肿瘤细胞的血管生成。通过qRT-PCR分析一系列血管生成相关分子的表达,蛋白质印迹和IFA。
■观察到DAB2IP的下调可以显着提高BCa细胞的克隆形成能力,降低他们对放疗和化疗药物的敏感性,增强他们的迁移和入侵能力,增加他们的干性特征。还注意到DAB2IP敲除或用来自DAB2IP缺陷的BCa细胞的条件培养基处理可以促进内皮细胞的管形成能力。同样,体内研究表明,与DAB2IP足够的肿瘤相比,siDAB2IPBCa细胞形成的肿瘤具有更高的肿瘤微血管密度(MVD)和更严重的氧缺乏。同时,DAB2IP的敲除抑制血管成熟并促进BCa组织中血管生成拟态(VM)的形成。下调STAT3可增强siDAB2IP细胞对IR的敏感性,同时伴有VEGF表达的降低。
■我们的数据支持,DAB2IP的丧失赋予BCa的放射抗性可能是由于缺氧增加,抑制血管成熟,促进VM形成。STAT3抑制可能是克服BCT中这种DAB2IP缺陷诱导的耐受性的潜在方法。
As a part of breast-conserving therapy (BCT), postoperative radiotherapy is one of the main means to improve the clinical efficacy of breast cancer (BCa). However, ionizing radiation (IR) may induce BCa cells to develop radioresistance, which causes tumor recurrence and metastasis after treatment. Recently, DOC-2/DAB2 interactive protein (
DAB2IP) has been reported often down-regulated in a variety of cancers and is related to tumor tolerance to radiotherapy. In this study, BCa cell lines were introduced to study how DAB2IP deficient influenced BCa cell radiosensitivity in vitro and in vivo and discuss the possible mechanism.
Small RNA interference system (siRNA) was employed to decrease
DAB2IP expression in two BCa cell lines, MDA-MB-231 and 4T1. Cells in response to IR or antineoplastics were detected by clone formation assay or MTT method, respectively. For in vivo studies, siDAB2IP or siControl cells were subcutaneously injected into the right flank of each female mouse. Sphere formation assay, soft agar colony anchoring assay and in vivo tumorigenesis assay were implemented to examine the stem cell-like features of BCa cells. Tube formation assay as well as immunofluorescence assay (IFA) were respectively applied to determine the angiogenesis of tumor cells in vitro and in vivo. The expression of a series of angiogenesis-related molecules was analyzed by qRT-PCR, western blot and IFA.
It was observed that the downregulation of
DAB2IP could significantly improve the clone formation ability of BCa cells, reduce their sensitivity to radiation and chemotherapy drugs, enhance their migration and invasion abilities and increase their stemness characteristics. It was also noted that either
DAB2IP-knocking down or treated with the conditioned medium from
DAB2IP-deficient BCa cells could promote the tube-forming ability of the endothelial cell. Similarly, in vivo studies showed that tumors developed from siDAB2IP BCa cells had higher tumor microvascular density (MVD) and more severe oxygen deficiency than that in DAB2IP- sufficient tumors. Meanwhile, Knock-down of DAB2IP inhibited vascular maturation and promoted the formation of vasculogenic mimicry (VM) in BCa tissues. Down-regulation of STAT3 could enhance siDAB2IP cells sensitivity to IR, accompanied by the decrease of VEGF expression.
Our data support that loss of DAB2IP confers radio-resistance of BCa could be due to increased hypoxia, inhibited vascular maturation and promoted VM formation. STAT3 inhibition could be a potential way to overcome such
DAB2IP-deficient induced tolerance in BCT.