Exploring Interactions Between Retinoid and Vitamin D Signaling in Salivary Gland Homeostasis and Cancer

摘要： 基底细胞来源的唾液癌和乳腺癌与患者预后差、癌症增加有关 复发和缺乏明确的治疗方法1.在两个分支器官中，细胞角蛋白5阳性(K5+)的基底细胞 在正常发育过程中对导管细胞的贡献2-4，最近的研究强调了在 K5+乳腺基底细胞起源和涎腺鳞状细胞5，6。在基底起源的乳腺肿瘤中，照射- 耐药肿瘤干细胞显示K5阳性1.维生素D和维生素A信号通路 与预防肿瘤发生有关7，8。在发育中的9个唾液腺中，我们和其他10个 最近证实，维甲酸受体(RAR)信号转导有助于K5+基底细胞的扩张， 在成人唾液腺中保守的现象11。在乳腺12和唾液腺13中，CSCs显示 ALDH是一种乙醛脱氢酶，能将维生素A氧化成视黄醇，是 维甲酸和通过RARs的信号。RAR信号转导通路与K5+细胞的关系 肿瘤的发生机制还有待探索。威尔士实验室的研究表明，维生素D的缺失 小鼠的受体(VDR)导致乳腺细胞增殖和肿瘤形成的增加，其中VDR活性 与K5+细胞命运转移有关。在人类群体研究中，低水平的维生素D与 癌症发病率和进展增加14-16。在下颌下唾液腺(SMG)中，祖细胞 人群中VDR 17呈高度阳性，在人类中，VDR定位于上皮细胞18；然而，维生素D 信号在唾液腺中还没有被专门研究过。我们将检验这一假说-- 激活VDR和RAR信号将减少SMG中K5+细胞的扩张和肿瘤的发生。 在这个提案中，我们将解决以下问题：1)VDR信号在K5+细胞中的作用是什么 在SMG中的扩展和分化？2)vdr信号与RARα信号是否协同负向 3)VdR信号的丢失与RARα的抑制是否有协同作用 信号增强K5+在SMG中的致瘤性4)VdR和RARα信号在K5- 应用维生素衍生物和小分子定向递送减少番茄Td阳性肿瘤细胞 致瘤细胞特性？确定RAR、α和VDR信号在负调节K5+中是否协同作用 在SmG中，在目标1中，我们将在Vdr基因敲除K5报告鼠中抑制RARα信号并跟踪 K5+基底细胞命运。在目标2中，我们将确定vdr和rarα通路是否协同调节K5+细胞 基底性涎腺肿瘤的扩张。这一目标将通过靶向药物抑制RARα来实现 DMBA诱导VDR基因敲除K5报告小鼠肿瘤发生的信号。此外，我们还将 用小分子检测K5-Td阳性番茄肿瘤细胞中VdR和RARα信号的激活 3D培养中的维生素衍生物会影响细胞分裂和运动。

Abstract: Basal cell derived salivary and breast cancers are correlated with poor patient prognosis, increased cancer reoccurrence and lack of definitive therapies 1. In both branching organs, cytokeratin 5-positive (K5+) basal cells contribute to ductal cells during normal development 2–4 and recent research has highlighted similarities in the K5+ basal cell origin of mammary and salivary squamous cells 5,6. In mammary tumors of basal origin, irradiation- resistant cancer stem cells (CSCs) demonstrate K5 positivity 1. Both Vitamin D and Vitamin A signaling pathways have been implicated in prevention of tumorigenesis 7,8. In the developing 9 salivary gland, we and others 10 have recently demonstrated that retinoic acid receptor (RAR) signaling contributes to expansion of K5+ basal cells, a phenomena conserved in the adult salivary gland 11. In both mammary 12 and salivary glands 13, CSCs display an upregulated level of ALDH, an aldehyde dehydrogenase that oxidizes Vitamin A to retinol, the precursor to retinoic acid and signaling through RARs. The relationship between RAR signaling and K5+ basal cells during tumorigenesis has yet to be explored. Work in the Welsh laboratory has shown that deletion of the Vitamin D receptor (VDR) in mice leads to increased cell proliferation and tumorigenesis in the breast 7 where VDR activity is linked to K5+ cell fate transition 4. In human population studies, low levels of Vitamin D are associated with increased cancer incidence and progression 14–16. In the submandibular salivary gland (SMG), progenitor cell populations are highly positive for VDR 17 and in humans, VDR localizes to epithelial cells 18; yet, Vitamin D signaling has not been specifically explored in the salivary gland. We will test the hypothesis that co- activating VDR and RAR signaling will reduce K5+ basal cell expansion and tumorigenesis in the SMG. In this proposal we will address the following questions: 1) What is the role of VDR signaling in K5+ basal cell expansion and differentiation in the SMG? 2) Does VDR signaling synergize with RARα signaling to negatively regulate K5+ basal cell expansion in the SMG? 3) Will loss of VDR signaling synergize with inhibition of RARα signaling to enhance K5+ tumorigenesis in the SMG? 4) Will activation of VDR and RARα signaling in K5- TdTomato-positive tumor cells using directed delivery of vitamin derivatives and small molecules decrease tumorigenic cell properties? To determine if RARα and VDR signaling synergize in negatively regulating the K5+ population in the SMG, in Aim 1 we will inhibit RARα signaling in a VDR knockout K5 reporter mouse and trace K5+ basal cell fate. In Aim 2, we will determine if VDR and RARα pathways synergize to regulate K5+ cell expansion in basal salivary tumors. This aim will be achieved with targeted pharmacological inhibition of RARα signaling in a VDR knockout K5 reporter mouse induced for tumorigenesis with DMBA. Additionally, we will determine if activation of VDR and RARα signaling in K5-TdTomato-positive tumor cells using small molecules and vitamin derivatives in 3D culture will affect cell division and motility.