Specific induction of lethal autophagy in triple-negative breast cancer cells

摘要 乳腺癌仍然是一种与高死亡率相关的严重的全球疾病。根据 年，美国疾病控制中心224,147名女性和2,125名男性被诊断患有乳腺癌 2012年。美国疾病控制与预防中心估计，在未来十年内，将有1970万人罹患乳腺癌。 目前对大多数乳腺癌的治疗往往依赖于干扰激素(ER、PR)或 这些细胞上有生长因子(HER2)受体。接受受体靶向治疗的患者有 预后良好，可长期存活。三阴性乳腺癌(TNBC)是由以下原因引起的癌症亚型 缺乏这三种受体的乳腺细胞的增殖，从而取消了这一选择 将靶向这些受体作为一种治疗策略。与其他类型的乳腺癌相比，个体 那些被诊断为TNBC的人治疗方案特别差，转移的几率更高 疾病，以及更短的生存时间。因此，TNBC细胞中的新分子和新途径可以有效地 迫切需要有针对性地治疗被诊断患有这种疾病的人。 除了它们较低的疗效外，目前用于TNBC的化疗药物还引起了许多 治疗药物的非靶标活动所产生的严重副作用。这些副作用 包括极度疲劳、不孕不育、恶心和心脏毒性。因此，至关重要的是，更具体地 确定了TNBC细胞中的靶点以进行治疗干预。最近，我们证明了LT-IIc，一种 细菌II型ADP-核糖化毒素(AR-TX)对TNBC细胞是致命的，但对非 转化的乳腺上皮细胞。初步观察表明LT-IIc对TNBC的致死作用 细胞是由于诱导了不可逆的自噬，这在正常的乳腺上皮细胞中是不存在的。我们会 使用LT-IIc作为探针来确定在TNBC细胞中特异性诱导的细胞毒机制。一个 将使用药理学、遗传学和荧光方法的组合来评估 神经节苷脂结合、ADP核糖化和细胞内cAMP对TNBC特异性的作用 细胞毒性。一组重组AR-TX嵌合体的可获得性将为这些实验提供便利 将TnBC-细胞毒LT-IIc的ADP核糖化和神经节苷脂结合亚基“混合搭配” 无细胞毒性的LT-IIb。选择性神经节苷脂表达和参与在TNBC特异性表达中的作用 细胞毒性反应，一个潜在的富有成效和未被探索的研究领域，将使用新的 TLC免疫印迹和质谱学。LT-IIc对肿瘤生长和转移的影响将是 使用小鼠TNBC肿瘤模型、体内成像和体外培养方法进行询问。 我们的近期目标是使用LT-IIc作为分子和细胞探测器，以发现新的和 可利用的TNBC特异性细胞毒途径。我们的长期目标是找出药理药剂 以这些途径为目标，并开发治疗TNBC的新的更安全的方法。

Abstract Breast cancer remains a serious global disease associated with high rates of mortality. According to The Centers for Disease Control, 224,147 women and 2,125 men in the U.S. were diagnosed with breast cancer in 2012. Over the next ten years, the CDC estimates that 19.7 million individuals will suffer from breast cancer. Current treatments for the majority of breast cancers often depend upon interfering with hormone (ER, PR) or growth factor (HER2) receptors on those cells. Individuals who receive receptor-targeted treatments have a good prognosis for long term survival. Triple-negative breast cancer (TNBC) is a subtype of cancer caused by proliferation of breast cells that are deficient in expression of those three receptors, thus abrogating the option of targeting of those receptors as a treatment strategy. In contrast to other types of breast cancers, individuals who are diagnosed with TNBC have exceptionally poor treatment options, a higher frequency of metastatic disease, and shorter survival times. Thus, new molecules and pathways in TNBC cells that can be efficiently targeted to treat individuals diagnosed with the disease are urgently needed. In addition to their lower efficacies, current chemotherapeutic agents for TNBC elicit a number of serious side effects that are engendered by off-target activities of the treatment agents. These side effects include overwhelming fatigue, infertility, nausea, and cardiac toxicity. Thus, it is critical that more specific targets in TNBC cells are identified for therapeutic intervention. Recently, we demonstrated that LT-IIc, a bacterial type II ADP-ribosylating toxin (AR-Tx), is lethal for TNBC cells, but has no lasting effects on non- transformed breast epithelial cells. Preliminary observations suggest that the lethal effects of LT-IIc for TNBC cells is due to induction of an irreversible autophagy that is absent in normal breast epithelial cells. We will employ LT-IIc as a PROBE to identify cytotoxic mechanisms that are specifically induced in TNBC cells. A combination of pharmacological, genetic, and fluorescent approaches will be employed to evaluate the contributions of ganglioside-engagement, ADP-ribosylation, and intracellular cAMP on TNBC-specific cytotoxicity. These experiments will be facilitated by the availability of a set of recombinant AR-Tx chimeras that “mix-and-match” the ADP-ribosylating and ganglioside-binding subunits of TNBC-cytotoxic LT-IIc and non-cytotoxic LT-IIb. The roles of preferential ganglioside expression and engagement in TNBC-specific cytotoxic responses, a potentially productive and unexplored area of research, will be evaluated using novel TLC immunoblotting and mass-spectroscopy. The effects of LT-IIc on tumor growth and metastasis will be interrogated using a mouse TNBC tumor model, in vivo imaging, and in vitro culture methods. Our immediate goal is to employ LT-IIc as a molecular and cellular PROBE to uncover new and exploitable TNBC-specific cytotoxic pathways. Our LONG TERM goal is to identify pharmacological agents that target those pathways and to develop new and safer approaches for treatment of TNBC.