Analysis of TLR agonist treated tumor cells

描述（由申请人提供）：最近，我们研究了LPS处理的4T1的生长减少，并发现LPS的作用依赖于Myd 88。令人惊讶的是，我们还发现，简单地降低Myd88表达足以减缓肿瘤细胞在体外和体内的生长。我们通过使用Myd88抑制肽抑制Myd88功能获得了类似的数据。用Myd88抑制剂治疗四种不同的鼠乳腺癌导致肿瘤细胞的体外生长速率降低。这些数据表明，通过Myd 88的信号传导对肿瘤生长很重要，并可能提供一种靶向乳腺癌的新方法。 这项研究的假设是Myd 88有助于乳腺癌的生长。为了解决这一假设，有三个具体目标：1）确定Myd88调节4T1肿瘤细胞中细胞周期进程和/或细胞凋亡抑制的程度，2）确定靶向Myd88在生长肿瘤中的治疗潜力，和3）确定HMGB1和HSP 60是否介导4T1肿瘤细胞中的Myd88依赖性信号传导。具体目的1将通过用Myd88抑制剂和Myd88特异性siRNA处理4T1细胞并确定生长抑制是否是由于细胞周期进展或细胞凋亡的抑制而实现。对于具体目标2，我们将在体内递送Myd 88抑制剂。将结合Myd 88靶向肿瘤的免疫原性和转移潜力监测肿瘤生长，以尝试将生长变化与抗肿瘤免疫力和疾病进展的改变相关联。这些研究将用4T1模型以及大鼠neu转基因小鼠进行，以确定在可移植和自发肿瘤模型中靶向Myd 88的治疗潜力。具体目标3将通过使用RNA干扰抑制4T1细胞中HMGB1和HSP 60的表达来实现，以确定（1）抑制这些DAMP的表达是否影响肿瘤细胞生长、细胞周期进展和/或凋亡，以及（2）抑制HMGB1或HSP 60是否重现抑制Myd 88的作用。 明确Myd 88在肿瘤中的作用可能会为癌症患者开发更有效的治疗策略。虽然Myd 88拮抗剂需要评估，但这已经是一个活跃的研究领域。研究人员正在探索Myd 88的变体，已经产生了抑制Myd 88功能的模拟物，最近使Myd 88-IRAK 4-IRAK 2死亡结构域复合物结晶，所有这些都可能有助于开发操纵Myd 88功能的新策略。因此，如果Myd 88拮抗剂可以帮助控制肿瘤进展，临床医生可能能够利用这些知识来帮助治疗癌症患者。阻断Mydd8功能的方法也可能与功能获得性突变导致癌症有关。最后，该项目的结果可能会更好地了解DAMP和PAMP如何影响肿瘤生长，并有助于产生旨在提高抗肿瘤免疫力的疗法。 公共卫生相关性：由于Myd 88的表达与肿瘤进展相关，因此人们不仅对确定Myd 88在肿瘤生长中的作用非常感兴趣，而且还对了解如何控制这种蛋白质的功能非常感兴趣。因此，目前对Myd 88的天然变体的研究和抑制Myd 88功能的合成模拟物的产生可能有助于开发操纵Myd 88功能并因此控制肿瘤生长的新策略。因此，该项目的结果可能为治疗癌症患者的新策略打开大门。

DESCRIPTION (provided by applicant): Recently, we investigated the decreased growth of LPS treated 4T1 and discovered that the effect of LPS was dependent upon Myd88. Surprisingly, we also found that simply decreasing Myd88 expression was sufficient to slow the growth of the tumor cells in vitro and in vivo. We obtained similar data by inhibiting Myd88 function using a Myd88 inhibitory peptide. Treatment of four different murine mammary carcinomas with the Myd88 inhibitor led to a decrease in the in vitro growth rate of the tumor cells. These data suggest that signaling through Myd88 is important for tumor growth and may provide a novel way to target breast cancer. The hypothesis of this study is that Myd88 contributes to the growth of breast cancer. To address this hypothesis, there are three specific aims: 1) determine the extent to which Myd88 regulates cell cycle progression and/or inhibition of apoptosis in 4T1 tumor cells, 2) determine the therapeutic potential of targeting Myd88 in growing tumors, and 3) determine whether HMGB1 and HSP60 mediate Myd88 dependent signaling in 4T1 tumor cells. Specific aim 1 will be accomplished by treating 4T1 cells with a Myd88 inhibitor and siRNA specific for Myd88 and determining whether inhibition of growth is due to inhibition of cell cycle progression or apoptosis. For specific aim 2, we will deliver the Myd88 inhibitor in vivo. Tumor growth will be monitored in conjunction with immunogenicity and metastatic potential of the Myd88 targeted tumors in an attempt to correlate changes in growth with alterations in anti-tumor immunity and disease progression. These studies will be conducted with the 4T1 model as well as rat neu transgenic mice in order to determine the therapeutic potential of targeting Myd88 in a transplantable and spontaneous tumor model. Specific aim 3 will be accomplished by inhibiting expression of HMGB1 and HSP60 in 4T1 cells using RNA interference to determine (1) if inhibiting expression of these DAMPs impacts tumor cell growth, cell cycle progression, and/or apoptosis and (2) if inhibiting HMGB1 or HSP60 recapitulates the effects of inhibiting Myd88. Defining the role of Myd88 in tumors may lead to the development of more effective treatment strategies for patients with cancer. Although Myd88 antagonists would need to be evaluated, this is already an active field of research. Investigators are exploring variants of Myd88, have generated mimics which inhibit Myd88 function, and recently crystallized the Myd88-IRAK4-IRAK2 death domain complex, all of which may assist in developing novel strategies to manipulate Myd88 function. Therefore, if Myd88 antagonists can help control tumor progression, clinicians may be able to utilize this knowledge to help treat patients with cancer. Methods to block Mydd8 function may also be relevant where gain-of-function mutations have been shown to contribute to cancer. Finally, results from this project may lead to a better understanding of how DAMPs and PAMPs impact tumor growth and assist in generating therapies aimed at increasing anti-tumor immunity. PUBLIC HEALTH RELEVANCE: Because Myd88 expression has been correlated with tumor progression, there is great interest in not only defining the role of Myd88 in tumor growth, but also in understanding how to control the function of this protein. As a result, current investigations of natural variants of Myd88 and the generation of synthetic mimics which inhibit Myd88 function may assist in developing novel strategies to manipulate Myd88 function and as a consequence control tumor growth. The results from this project therefore may open the door to new strategies for the treatment of patients with cancer.