NSBP1调控食管癌耐药的作用机制及其临床意义研究

Esophageal cancer is the sixth most common cause of cancer-related deaths in the world and the prognosis is generally poor. China alone accounts for approximately half of all new esophageal cancer cases per year, with esophageal squamous cell carcinoma (ESCC) being the predominant histologic subtype. Tumor chemoresistance is significantly contributed to the high lethality of esophageal cancer. Illustrating the molecular mechanisms that promote chemoresistance of cancer cells is therefore essential to tackle this highly aggressive disease. ..Although other researchers had conducted gene profiling studies on ESCC to identify diagnostic, prognostic or predictive biomarkers, there is as yet little information and functional validation of chemoresistant genes. We have established human ESCC cells of acquired chemoresistance to 5-FU (designated as FR cells), which can be used to identify important regulators of cancer chemoresistance. Gene profiling identified NSBP1 as one of the top upregulated genes in highly chemoresistant ESCC cells. However, the clinical relevance and function of NSBP1 in esophageal cancer has not been documented previously. ..Firstly, we explored the clinicopathological significance of NSBP1 by studying the expression level of NSBP1 in primary esophageal cancer and paired normal tissue. By analyzing 50 primary tumor and paired normal tissues, our results indicated that clinical ESCC tumor tissues expressed higher level of NSBP1 compared with corresponding normal esophageal tissue. Notably, we found that the patients with high NSBP1 expression had shorter survival than patients with low NSBP1 expression...Furthermore, our preliminary data showed that ectopic overexpression of NSBP1 significantly enhanced chemoresistance of esophageal cancer in vitro and in vivo. Further comparison of gene profiling and Ingenuity Pathyway Analysis(IPA) analysis of NSBP1-overexpressing ESCC cells with control cells suggested that the collaboration of protein phosphatase 2 regulatory subunit B delta (PPP2R2D) and Syndecan Binding Protein (SDCBP), may mediate the function of NSBP1 in ESCC chemoresistance. Western Blot results illustrated that forced expression of NSBP1 increased the expression level of SDCBP, decreased the expression of PPP2R2D, thereby activating AKT pathway. On the contrary, transfection with siRNA against NSBP1 decreased the expression level of SDCBP, increased the expression of PPP2R2D. Notably, we found that blockade of AKT signaling with the AKT inhibitor, LY294002, abrogated the stimulating effects of NSBP1 on chemoresistance. ..In this study, we hypothesize that NSBP1 could serve a good diagnostic and prognostic biomarker; NSBP1 could enhance chemoresistance of ESCC via the PPP2R2D/SDCBP-AKT signaling axis. This regulatory mechanism has not been reported previously. ..To further validate this hypothesis, we proposed to carry more experiments with the aims:...1..By using gain and loss-of-function studies, further investigate whether NSBP1 promotes chemoresistance of esophageal cancer cells in vitro and in vivo..2..To study the role of PPP2R2D/SDCBP-AKT signaling axis in mediating the effects of NSBP1 on esophageal cancer cell chemoresistance, and to study the molecular mechanism by which NSBP1 regulates PPP2R2D and SDCBP..3..To evaluate the potential of NSBP1 as diagnostic and prognostic marker in esophageal cancer, in particular, the correlation of NSBP1 expression with chemotherapy response in esophageal cancer treatment...The long-term impact is to obtain a better understanding of the detailed molecular functions of this intriguing oncogene in cancer. Deciphering the role and mechanism of NSBP1 in promoting drug resistance will also shed light on the treatment of esophageal cancer.

肿瘤细胞对化疗药物耐药，是造成食管癌生存率低的主要原因。目前，肿瘤的耐药机制不明确，缺乏有效预测化疗疗效的分子标志物及相应的逆转耐药的有效措施。前期工作中，我们建立了多条对5-FU高度耐药的食管癌细胞株，通过基因芯片对比耐药细胞和亲本细胞，发现NSBP1在耐药细胞中有显著上调。然而，NSBP1在调控肿瘤5-FU耐药方面的生物功能和作用机制尚不可知。我们发现，NSBP1在肿瘤组织中明显高表达, 并且与患者生存期显著负相关。体内体外实验证实，过表达NSBP1可以明显增强食管癌细胞的耐药能力。通过运用基因芯片、生物信息学IPA分析和一系列实验验证，我们发现NSBP1可能通过促进SDCBP和抑制PPP2R2D而激活AKT通路，最终导致5-FU耐药。我们拟从生物学功能、分子调控机制、潜在临床意义三个方面剖析NSBP1在肿瘤耐药中的作用。本项目可为食管癌肿瘤耐药机制提供新方向，为临床诊疗提供新线索。

肿瘤细胞对化疗药物的耐药，是造成食管癌，这一恶性肿瘤生存率低的主要原因。然而，肿瘤的耐药机制非常复杂，分子信号调控机制仍不明确， 缺乏有效的预测药物敏感性和药物反应的分子标志物及相应的耐药逆转的有效的措施。 前期工作中，我们建立了多条高度耐药食管癌细胞株，通过基因芯片对比亲本细胞和耐药细胞，发现NSBP1在耐药细胞中有高于5倍的显著上调。然而，NSBP1在调控食管癌肿瘤耐药方面的生物功能和作用机制尚不可知。.我们发现NSBP1在肿瘤组织中明显高表达, 并且与患者生存率显著负相关， 然而NSBP1与患者的耐药的关系未知。最新的体内体外研究显示，过表达NSBP1可以显著地增加肿瘤细胞对化疗药物5-FU的耐药性，反之， 敲低NSBP1可以明显地增加肿瘤细胞细胞对药物的敏感性。我们基于人源化小鼠模型（PDX），进一步构建耐药和敏感的PDX模型，我们的结果显示，NSBP1的表达在耐药的瘤体中显著比敏感的瘤体增多。接下来，我们进一步研究其下游分子机制，结果提示NSBP1可能通过SDCBP激活AKT信号通路。 另外，我们从胞外角度，运用蛋白芯片分析对比NSBP1过表达以及对照细胞的条件培养基，寻找NSBP1在肿瘤微环境中的作用靶蛋白， 结果提示，NSBP1可能通过分泌ICAM-3影响肿瘤微环境中的成纤维细胞； 接下来，我们也运用ChIP-seq需要NSBP1的作用蛋白，结果提示，NSBP1可以通过影响ATF3的转录，进而再影响SDCBP的转录从而影响其功能，最后，我们基于SDCBP的晶体结构进行虚拟筛选，选出可以同时靶向SDCBP,抑制肿瘤生长和对耐药有作用的小分子抑制剂。.总之，我们从潜在临床意义，生物学功能和分子机制三个方面剖析NSBP1在肿瘤耐药中的作用。本项目能为食管癌肿瘤发生、发展、特别是耐药机制提供新的解说和思路，为临床治疗提供新的靶点和契机。