Somatostatin Receptor Subtype 5 Regulation of Islet Neoplasia

摘要：在当前的R-01项目资助期间，我们的团队取得了4项与人类和 小鼠生长抑素受体亚型5（hSSTR 5，mSSTR 5）和胰十二指肠同源框-1（PDX-1） 对于患有胰岛瘤形成（INeo）的患者来说是高度可转化的：1.我们的SSTR 5-/-小鼠发生INeo 与高胰岛素血症、低血糖和PDX-1过表达相关，表明mSSTR 5调节 胰岛素分泌和INeo通过PDX-1; 2. PDX-1调控INeo细胞增殖和iv PDX-1 shRNA 在转移性胰岛素瘤小鼠模型中，PDX-1治疗可预防低血糖死亡，这表明PDX-1是一种治疗低血糖的药物。 INeo的分子靶标; 3.我们发现了一个hSSTR 5基因的SNP，在C 在约50%的人INeo肿瘤中，末端位置335（hSSTR 5 P335 L）。初步的体外数据显示， hSSTR 5 P335 L功能低下导致PDX-1过表达和缺乏对SSTR 5类似物的应答 INeo细胞中的PRL-1980，表明基因组hSSTR 5变异预测对治疗的响应; 4.我们的老鼠胰岛素 可以使用由PDX-1激活的启动子-胸苷激酶（RIP-TK）载体和F18-FHBG前药 对小鼠INeo肿瘤中的PDX-1表达进行成像，并可用于跟踪对治疗的反应。 假设：a）hSSTR 5通过PDX-1调节胰岛素和INeo，B）hSSTR 5 P335 L是功能低下的受体 具有改变的PDX-1对INeo的调节，和c）hSSTR 5的基因组变异和 hSSTR 5、hSSTR 5 P335 L和PDX-1预测对靶向疗法的应答。具体目标1：确定 a）INeo患者中hSSTR 5基因组变异的分布是否可以使用TaqMan等位基因验证 区分测定和B）INeo的hSSTR 5、hSSTR 5 P335 L和/或PDX-1表达的变化 可以使用QPCR、蛋白质印迹和量子点（Q Dot）去卷积成像来验证样本， 图像分析。具体目的2：确定a）hSSTR 5是否调节胰岛素和INeo细胞增殖 通过PDX-1，B）hSSTR 5 P335 L是一种功能减退的受体，具有改变的PDX-1对INeo的调节，和c） hSSTR 5基因组变异和hSSTR 5、hSSTR 5 P335 L和PDX-1表达水平的变异预测 i）TC-6 hSSTR 5 SCID小鼠中对SSTR 5特异性类似物PRL-1980与PDX-1 shRNA治疗的反应 模型，ii）TC-6 hSSTR 5 P335 L SCID小鼠模型，iii）TC-6SSTR 5 KD（敲低）SCID小鼠模型，iv）hSSTR 5 敲入小鼠，v）hSSTR 5 P335 L敲入小鼠和vi）SSTR 5-/-小鼠。具体目标3：确定是否 a）使用大鼠胰岛素启动子-胸苷激酶（RIP-TK）在INeo中检测PDX-1表达的成像剂 来自aim 2的小鼠模型可以使用18F-FHBG进行microPET成像和/或双标记 用于光学成像和断层摄影术的类似物，B）可以使用 RIP-TK/ 18F-FHBG microPET成像，并且依赖于PDX-1表达水平，以及 1980-Cu 64成像可以区分SSTR 5阳性和SSTR 5阴性INeo肿瘤， 证实PDX-1在体内治疗反应中的作用。

ABSTRACT: During the funding of the current R-01, our team made 4 discoveries related to human and mouse somatostatin receptor subtype five (hSSTR5, mSSTR5) and pancreatic duodenal homeobox-1 (PDX-1) that are highly translatable for patients suffering with islet neoplasia (INeo): 1. our SSTR5-/- mice develop INeo associated with hyperinsulinemia, hypoglycemia and PDX-1 overexpression, suggesting mSSTR5 regulates both insulin secretion and INeo via PDX-1; 2. PDX-1 regulates INeo cell proliferation and iv PDX-1 shRNA therapy prevents hypoglycemic death in a metastatic insulinoma mouse model, suggesting PDX-1 is a molecular target for INeo; 3. we found a germline hSSTR5 SNP with a proline to leucine substitution at C terminal position 335 (hSSTR5 P335L) in ~50% of human INeo tumors. Preliminary in vitro data reveal that hSSTR5 P335L is hypofunctional resulting in PDX-1 overexpression and lack of response to SSTR5 analogue PRL-1980 in INeo cells, suggesting genomic hSSTR5 variation predicts response to therapy; 4. our rat insulin promoter-thymidine kinase (RIP-TK) vector, which is activated by PDX-1, and F18-FHBG prodrug can be used to image PDX-1 expression in INeo tumors in mice and could be used to track response to therapy. Hypotheses: a) hSSTR5 regulates insulin and INeo via PDX-1, b) hSSTR5 P335L is a hypofunctional receptor with altered PDX-1 regulation of INeo, and c) genomic variation of hSSTR5 and variations in expression of hSSTR5, hSSTR5 P335L and PDX-1 predict response to targeted therapies. Specific Aim 1: to determine whether a) distribution of hSSTR5 genomic variation in INeo patients can be validated using TaqMan Allelic Discrimination assay and b) variations in expression of hSSTR5, hSSTR5 P335L and/or PDX-1 of INeo specimens can be validated using QPCR, Western blot and Quantum Dot (Q Dot) deconvolution imaging and image analysis. Specific aim 2: to determine whether a) hSSTR5 regulates insulin and INeo cell proliferation via PDX-1, b) hSSTR5 P335L is a hypofunctional receptor with altered PDX-1 regulation of INeo, and c) hSSTR5 genomic variation and variations in hSSTR5, hSSTR5 P335L and PDX-1 expression levels predict response to SSTR5-specific analogue PRL-1980 vs PDX-1 shRNA therapies in i) βTC-6hSSTR5 SCID mouse model, ii) βTC-6hSSTR5 P335L SCID mouse model, iii) βTC-6SSTR5KD (Knock Down) SCID mouse model, iv) hSSTR5 knock-in mice, v) hSSTR5 P335L knock-in mice and vi) SSTR5-/- mice. Specific Aim 3: to determine whether a) imaging agents to detect PDX-1 expression using rat insulin promoter-thymidine kinase (RIP-TK) in INeo mouse models from aim 2 can be validated using 18F-FHBG for microPET imaging and/or dual labeled analogues for optical imaging and tomography, b) the response to targeted therapies can be studied using RIP-TK/ 18F-FHBG microPET imaging and is dependent upon PDX-1 expression levels and c) microPET PRL- 1980-Cu64 imaging can differentiate between SSTR5 positive and SSTR5 negative INeo tumors and will confirm the role of PDX-1 in therapeutic response in vivo.