Targeting Stat1 and Stat3 to Reverse Radioresistance in Head and Neck Cancer

 描述(申请人提供)：分割放射加电离辐射(IR)是HNSCC治疗的主要手段，但在三分之二的病例中失败，很大程度上是由于IR耐药性的产生。需要对IR抗性的分子基础有新的见解，以开发能够克服它的试剂。最近，从对IR敏感的HNSCC细胞系SCC-61开始，通过连续移植和照射异种移植瘤，培育出了一种耐IR的HNSCC细胞系NU-61。比较最初和最终细胞系的基因表达谱，确定25个干扰素/STAT1途径特征基因是获得性辐射抵抗的候选基因。在NU-61中沉默STAT1使这些基因的表达减少并使其对IR敏感，而在对辐射敏感的SCC-61亲本细胞中过表达STAT1则增加了这些基因的表达并将其转化为IR抗性。我们证明了同一组特征基因在另一个耐IR细胞系UM-SCC-17B的异种移植瘤中上调，这进一步支持了干扰素/STAT1途径可能在HNSCC的辐射抗性中起关键作用和值得靶向的假设。HNSCC的IR抵抗也与STAT3的激活有关，这是通过增加EGFR或LIF信号来实现的。为了开发逆转辐射抗性的新策略，我们团队使用基于计算机的对接和铅化合物优化策略来鉴定C188-9，一种有效的、无毒的、口服生物可用的STAT3和STAT1双重抑制剂。C188-9对耐IR的HNSCC细胞株UM-SCC-17B的移植瘤生长有明显的抑制作用。对这些异种移植瘤的RNA-SEQ分析表明，C188-9调控91个由STAT3和/或STAT1直接调控的基因，包括干扰素/STAT1信号集中的25个基因中的23个，其中许多已知也受到STAT3的共同调控。在这项建议中，我们假设STAT1和STAT3参与了IR抵抗，并且使用双活性STAT1/3抑制剂C188-9作为IR的佐剂将比单独使用IR更有效地缩小HNSCC肿瘤移植瘤。我们已经概述了两个有针对性的具体目标来审问这些假说。目的1研究IR加或不加C188-9对耐IR的HNSCC细胞(UM-SCC-17B、SCC-35和NU-61)和IR敏感的HNSCC细胞(SCC-61、SCC-15、SCC-9)的生长、STAT1和STAT3活性以及干扰素/STAT1信号基因表达的影响。在目标2中，我们将在体内检测IR加或不加C188-9对耐IR HNSCC移植瘤生长、STAT1和STAT3活性以及干扰素/STAT1信号基因表达的影响。IR与无毒剂的结合，如C188-9，分子靶向一个或多个辐射抵抗的关键介体，尚未到达临床。这个项目的成功完成将使我们能够 评估STAT1和STAT3的双重靶向是否是克服HNSCC放射抵抗的潜在有用的策略。如果成功，这些研究将为C188-9作为IR佐剂在HNSCC中进行临床前试验奠定基础，以减少IR耐药性的出现并提高治愈率。

 DESCRIPTION (provided by applicant): Fractionated radiotherapy with ionizing radiation (IR) is a mainstay of HNSCC treatment but fails in two-thirds of cases due, in large part, to development of IR resistance. New insights into the molecular basis for IR resistance are needed to develop agents capable of overcoming it. Recently an IR-resistant HNSCC cell line, Nu-61, was developed by serial transplantation and irradiation of xenograft tumors starting with the IR-sensitive HNSCC cell line, SCC-61. Comparison of the gene expression profiles of the initial and final cell lines identified 25 IFN/Stat1 pathway signature genes as candidates for acquired radioresistance. Silencing Stat1 in Nu-61 decreased expression of these genes and rendered these cells IR sensitive, whereas overexpression of Stat1 in the radiosensitive SCC-61 parental cells increased expression of these genes and converted them to IR resistant. We demonstrated that the same set of signature genes is upregulated in xenografts from another IR- resistant cell line, UM-SCC-17B, which further supports the hypothesis that the IFN/Stat1 pathway may be critical for radioresistance and worth targeting in HNSCC. IR resistance in HNSCC also has been linked to Stat3 activation via increased EGFR or LIF-signaling. To develop new strategies for reversing radioresistance, our group used computer-based docking and lead-compound optimization strategies to identify C188-9, a potent, non-toxic, and orally bioavailable dual inhibitor of Stat3 and Stat1. C188-9 potently inhibited growth of tumor xenografts of the IR-resistant HNSCC cell line, UM-SCC-17B. RNA-seq analysis of these xenografts revealed that C188-9 modulated 91 genes regulated directly by Stat3 and/or Stat1 including 23 of the 25 genes in the IFN/Stat1 signature set, many of which also are known to be co-regulated by Stat3. In this proposal, we hypothesize that Stat1 and Stat3 contribute to IR resistance and that use of the dual active Stat1/3 inhibitor, C188-9, as an adjuvant with IR will be more effective at shrinking HNSCC tumor xenografts than IR alone. We have outlined two focused Specific Aims to interrogate these hypotheses. In Aim 1, we will determine the effect in vitro of IR with or without C188-9 on the growth, Stat1 and Stat3 activity, and expression of IFN/Stat1 signature genes in both IR-resistant HNSCC cell lines (UM-SCC-17B, SCC-35, and Nu-61) and IR-sensitive HNSCC cell lines (SCC-61, SCC-15, SCC-9). In Aim 2, we will determine the effect in vivo of IR with or without C188-9 on growth, Stat1 and Stat3 activity, and expression of IFN/Stat1 signature genes in IR-resistant HNSCC xenografts. The combination of IR with a non-toxic agent, such as C188-9, that molecularly targets one or more critical mediator of radioresistance has yet to reach the clinic. Successful completion of this project will allow us to assess whether dual targeting of Stat1 and 3 is a potentially useful strategy for overcoming radioresistance in HNSCC. If successful, these studies will form the preclinical basis for performing clinical trials of C188-9 as an adjuvant to IR in HNSCC to reduce emergence of IR resistance and improve cure rates.