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

摘要 乳腺癌仍然是一种严重的全球性疾病，死亡率很高。根据 根据美国疾病控制中心的数据，2009年，美国有224，147名女性和2，125名男性被诊断患有乳腺癌。 2012.在接下来的十年里，CDC估计将有1970万人患有乳腺癌。 目前大多数乳腺癌的治疗通常依赖于干扰激素（ER，PR）或激素受体（ER）。 生长因子（HER 2）受体。接受受体靶向治疗的个体 长期生存预后良好。三阴性乳腺癌（TNBC）是由乳腺癌引起的癌症亚型， 这三种受体表达缺陷的乳腺细胞增殖，从而取消了选择 将这些受体作为治疗策略。与其他类型的乳腺癌相比， 被诊断患有TNBC的人的治疗选择非常差，转移性肿瘤的发生频率更高， 疾病和更短的生存时间。因此，TNBC细胞中的新分子和途径，可以有效地 迫切需要有针对性地治疗被诊断患有该疾病的个体。 除了其较低的功效之外，目前用于TNBC的化疗剂引起了许多不良反应。 由治疗剂的脱靶活性引起的严重副作用。这些副作用 包括极度疲劳、不育、恶心和心脏毒性。因此，至关重要的是， 鉴定TNBC细胞中的靶标用于治疗干预。最近，我们证明了LT-IIc， 细菌II型ADP-核糖基化毒素（AR-Tx）对TNBC细胞是致命的，但对非TNBC细胞没有持久的影响。 转化的乳腺上皮细胞。初步观察表明，LT-IIc对TNBC的致死作用 细胞的凋亡是由于诱导不可逆的自噬，这在正常乳腺上皮细胞中是不存在的。我们将 使用LT-IIc作为探针，以鉴定在TNBC细胞中特异性诱导的细胞毒性机制。一 将采用药理学、遗传学和荧光方法的组合来评估 神经节苷脂结合、ADP-核糖基化和细胞内cAMP对TNBC特异性 细胞毒这些实验将通过一组重组AR-Tx嵌合体的可用性来促进 “混合匹配”TNBC细胞毒性LT-IIc的ADP核糖基化和神经节苷脂结合亚基， 无细胞毒性LT-IIb。神经节苷脂的优先表达和参与在TNBC特异性肿瘤细胞中的作用 细胞毒性反应，一个潜在的生产力和未开发的研究领域，将评估使用新的 TLC免疫印迹和质谱。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。 我们的近期目标是采用LT-IIc作为分子和细胞探针， 可利用的TNBC特异性细胞毒性途径。我们的长期目标是确定药物 针对这些途径，并开发新的和更安全的方法来治疗TNBC。

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.