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

 描述（由申请人提供）：电离辐射（IR）的分次放疗是HNSCC治疗的主要方法，但在三分之二的病例中失败，这在很大程度上是由于IR抵抗的发展。最近，通过从IR敏感的HNSCC细胞系SCC-61开始的异种移植肿瘤的连续移植和照射，开发了IR抗性HNSCC细胞系Nu-61。初始和最终细胞系的基因表达谱的比较确定了25个IFN/Stat 1途径标记基因作为获得性辐射抗性的候选基因。Nu-61中Stat 1的沉默降低了这些基因的表达并使这些细胞对IR敏感，而放射敏感性SCC-61亲本细胞中Stat 1的过表达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来自另一种IR抗性细胞系UM-SCC-17 B的异种移植物中上调，这进一步支持了IFN/Stat 1途径可能对辐射抗性至关重要并且值得在HNSCC中靶向的假设。HNSCC中的IR抗性还通过增加的EGFR或LIF信号传导与Stat 3活化相关。为了开发逆转辐射抗性的新策略，我们的研究小组使用基于计算机的对接和先导化合物优化策略来鉴定C188-9，这是一种强效、无毒、口服生物可利用的Stat 3和Stat 1双重抑制剂。C188-9有效抑制IR抗性HNSCC细胞系UM-SCC-17 B的肿瘤异种移植物的生长。对这些异种移植物的RNA测序分析显示，C188-9调节了由Stat 3和/或Stat 1直接调节的91个基因，其中包括IFN/Stat 1特征集中25个基因中的23个，其中许多基因也已知受到Stat 3的共调节。在这个提议中，我们假设Stat 1和Stat 3有助于IR抗性，并且使用双重活性Stat 1/3抑制剂C188-9作为IR的佐剂将比单独IR更有效地收缩HNSCC肿瘤异种移植物。我们已经概述了两个有针对性的具体目标来质疑这些假设。在目标1中，我们将在体外确定IR联合或不联合C188-9对IR耐药HNSCC细胞系（UM-SCC-17 B、SCC-35和Nu-61）和IR敏感HNSCC细胞系（SCC-61、SCC-15、SCC-9）的生长、Stat 1和Stat 3活性以及IFN/Stat 1特征基因表达的影响。在目标2中，我们将确定在体内IR与或不与C188-9对生长，Stat 1和Stat 3活性，以及在IR耐药HNSCC异种移植物中IFN/Stat 1特征基因的表达的影响。IR与分子靶向一种或多种辐射抗性关键介质的无毒剂如C188-9的组合尚未进入临床。这个项目的成功完成将使我们 评估Stat 1和Stat 3的双重靶向是否是克服HNSCC放射抗性的潜在有用策略。如果成功，这些研究将为C188-9在HNSCC中作为IR佐剂进行临床试验以减少IR耐药性的出现并提高治愈率奠定临床前基础。

 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.