Somatostatin Receptor Subtype 5 Regulation of Islet Neoplasia

摘要：在当前R-01的资助期间，我们团队有4项与人类和人类有关的发现 小鼠生长抑素受体亚型5(hSSTR5、mSSTR5)和胰腺十二指肠同源盒-1(PDX-1) 对于患有胰岛肿瘤(Ineo)的患者来说是高度可翻译的：1.我们的SSTR5-/-小鼠患上了Ineo 与高胰岛素血症、低血糖和PDX-1过度表达有关，提示mSSTR5调节 胰岛素分泌和Ineo均通过PDX-1实现；2.PDX-1调节Ineo细胞增殖和静脉注射PDX-1 shRNA 治疗预防转移性胰岛素瘤小鼠模型中的低血糖死亡，提示PDX-1是一种 Ineo的分子靶点；3.我们发现了一个生殖系hSSTR5 SNP，在C位有一个由Pro取代为亮氨酸的SNP 末端位置335(HSSTR5 P335L)在约50%的人眼部肿瘤中存在。初步的体外数据显示， HSSTR5 P335L功能低下导致PDX-1过度表达，对SSTR5类似物缺乏反应 在Ineo细胞中的PRL-1980，表明基因组hSSTR5变异预测治疗反应；4.我们的大鼠胰岛素 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 目的：对PDX-1在小鼠Ineo肿瘤中的表达进行成像，并可用于追踪治疗反应。 假设：a)hSSTR5通过PDX-1调节胰岛素和Ineo，b)hSSTR5 P335L是一个功能低下的受体 C)hSSTR5的基因组变异和hSSTR5基因表达的变异 HSSTR5、hSSTR5 P335L和PDX-1预测靶向治疗的反应。具体目标1：确定 是否可以使用TaqMan等位基因验证hSSTR5基因组变异在Ineo患者中的分布 Ineo细胞hSSTR5、hSSTR5 P335L和/或PDX-1基因表达的差异 标本可以使用QPCR、Western印迹和量子点(Q Dot)去卷积成像进行验证，并 图像分析。特定目的2：确定a)hSSTR5是否调节胰岛素和Ineo细胞的增殖 通过PDX-1，b)hSSTR5 P335L是一种功能低下的受体，但PDX-1对Ineo的调节发生了变化，以及c) HSSTR5基因组变异和hSSTR5、hSSTR5 P335L和PDX-1表达水平的变异可预测 SCID小鼠对SSTR5特异性类似物PRL-1980与PDX-1 shRNA治疗的反应 模型，二)&#946；TC-6hSSTR5 P335L SCID小鼠模型，三)&#946；TC-6SSTR5KD(敲击)SCID小鼠模型，四)hSSTR5 敲除小鼠，v)hSSTR5 P335L敲入小鼠和vi)SSTR5-/-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表达的显像剂 可以使用18F-FHBG对来自AIM 2的小鼠模型进行microPET成像和/或双标记 光学成像和断层扫描的类似物，b)对靶向治疗的反应可以使用 RIP-TK/18F-FHBG microPET成像，取决于PDX-1的表达水平和c)microPET PRL- 1980年-Cu64成像可以区分SSTR5阳性和SSTR5阴性的新生血管肿瘤和Will 确认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.