Inhibition of Cancer stem cell characterisitcs in Pancreatic Cancer

描述（由申请人提供）： 摘要肿瘤干细胞（CSCs）是胰腺癌发生、发展和化疗失败的重要原因。在PanIN病变向转移性肿瘤的进展期间，在胰腺癌中发生异常的刺猬信号传导，这表明声波刺猬（Shh）信号传导可能是导致胰腺癌中未分化前体细胞积累的早期事件。该项目基于萝卜硫素（SFN），一种来自十字花科蔬菜的天然化合物，可用于预防和/或治疗人类胰腺癌的前提。不幸的是，SFN抑制胰腺癌细胞和CSC的生长和转移并诱导凋亡的细胞内机制从未被研究过。因此，本申请的主要目的是检查莱菔硫烷抑制胰腺CSC特征的分子机制，并评估莱菔硫烷通过靶向CSC对胰腺癌的化学预防/治疗潜力。这些研究将为我们提供新的知识，指导我们制定通过靶向CSC预防人类胰腺癌的策略。我们最近已经证明，人CD 133 + CD 44 + CD 24 +ESA+ CSC表达Nanog、Notch-1和Oct-4，并且在小鼠中具有高度致瘤性。我们的初步数据表明：（i）SFN抑制胰腺CSC的自我更新能力;（ii）SFN抑制多能性维持因子的表达（Nanog、Oct-4和Sox-2），上皮间质（EMT）标志物（Zeb-1、Snail、Slug）、Bcl-2和XIAP;（iii）SFN抑制Smo、Ptch以及效应分子Gli 1和Gli 2的表达，提示Shh通路在胰腺癌中的临床意义;（iv）Shh通路的激活或shRNA对Gli 1和Gli 2表达的抑制可阻断SFN的抑制作用，提示萝卜硫素的这些作用是通过Shh通路介导的;和（v）SFN抑制从人原发性肿瘤和KrasG 12 D小鼠的胰腺分离的CSC（具有高Nanog的CD 133 + CD 44 + CD 24 +ESA+细胞）的自我更新能力。基于我们的初步研究结果，我们假设萝卜硫素单独或与标准化疗药物联合通过调节多能性促进因子和抑制音刺猬途径来抑制胰腺癌干细胞特征。上述假设将通过以下四个具体目标进行检验：目标1。研究莱菔硫烷抑制人胰腺癌干细胞特性和体外致敏耐药肿瘤干细胞的分子机制。目标二。研究萝卜硫素是否通过Sonic Hedgehog通路抑制胰腺癌干细胞的自我更新能力、迁移、侵袭和上皮间质转化，并进一步研究Shh通路在肿瘤-间质相互作用中的作用。目标3：使用KrasG 12 D转基因小鼠模型检查口服给予莱菔硫烷对胰腺癌发生的影响。目标4。验证口服给予莱菔硫烷是否通过抑制音刺猬途径有效抑制胰腺癌干细胞特征和肿瘤生长，并在NOD/SCID/IL 2 R γ小鼠模型中检查莱菔硫烷与标准化疗药物的相互作用。我们的研究是非常新颖和重要的，因为它们具有预后相关性，并通过靶向CSC抑制胰腺癌的发生和进展。

DESCRIPTION (provided by applicant): ABSTRACT Cancer stem cells (CSCs) have been proposed recently to be the cause cancer initiation, progression and chemotherapy failure in pancreatic cancer. Abberant hedgehog signaling occurs in pancreatic cancer during the progression of PanIN lesions to metastatic tumors, suggesting that sonic hedgehog (Shh) signaling may be an early event leading to accumulation of undifferentiated precursor cells in pancreatic cancer. This project is based on the premise that sulforaphane (SFN), a natural compound from the cruciferous vegetables, can be used for prevention and/or treatment of human pancreatic cancer. Unfortunately, the intracellular mechanisms by which SFN inhibits growth and metastasis, and induces apoptosis in pancreatic cancer cells and CSCs have never been examined. Thus, the main objective of this application is to examine the molecular mechanisms by which sulforaphane inhibits pancreatic CSC characteristics, and assess the chemopreventive / therapeutic potential of sulforaphane against pancreatic cancer by targeting CSCs. These studies will empower us with new knowledge that will guide us in formulating strategies for prevention of human pancreatic cancer by targeting CSCs. We have recently demonstrated that human CD133+CD44+CD24+ESA+ CSCs express Nanog, Notch-1 and Oct-4 and are highly tumorigenic in mice. Our preliminary data indicate that: (i) SFN inhibits self-renewal capacity of pancreatic CSCs; (ii) SFN inhibits the expression of pluripotency maintaining factors (Nanog, Oct-4 and Sox-2), epithelial mesenchymal (EMT) markers (Zeb-1, Snail, Slug), Bcl-2 and XIAP in pancreatic CSCs; (iii) SFN inhibits the expression of Smo, Ptch as well as effector molecule Gli 1 and 2, suggesting the clinical significance of Shh pathway in pancreatic cancer; (iv) enforced activation of Shh pathway or inhibition of Gli1 plus Gli2 expression by shRNA blocked the inhibitory effects of SFN, suggesting these effects of sulforaphane are mediated through Shh pathway; and (v) SFN inhibits self-renewal capacity of CSCs (CD133+CD44+CD24+ESA+ cells with high Nanog) isolated from pancreas of human primary tumors and KrasG12D mice. Based on our preliminary findings, we hypothesize that sulforaphane alone or in combination with standard chemotherapeutic drugs inhibit pancreatic cancer stem cell characteristics by regulating pluripotency promoting factors and inhibiting the sonic hedgehog pathway. The above hypothesis will be tested by the following four specific aims: Aim 1. To examine the molecular mechanisms by which sulforaphane inhibits human pancreatic cancer stem cell characteristics and sensitizes drug- resistant cancer stem cells in vitro. Aim 2. To examine whether sulforaphane inhibits self-renewal capacity, migration, invasion and epithelial mesenchymal transition of pancreatic cancer stem cells through sonic hedgehog pathway and further to examine the involvement of Shh pathway in tumor-stroma interaction. Aim 3. To examine the effects of oral administration of sulforaphane on pancreatic carcinogenesis using a KrasG12D transgenic mouse model. Aim 4. To validate whether oral administration of sulforaphane is effective in inhibiting pancreatic cancer stem cell characteristics and tumor growth by inhibiting sonic hedgehog pathway, and to examine the interactive effects of sulforaphane with standard chemotherapeutic drugs in a NOD/SCID/IL2Rgammanull mouse model. Our studies are very novel and significant because they have prognostic relevance and inhibit pancreatic cancer initiation and progression by targeting CSCs.