Role of P4HA1 in Endocrine Resistance of Breast Cancer

摘要： 他莫昔芬是最古老和最广泛使用的选择性雌激素受体调节剂。氟维司群是一 选择性雌激素受体下调剂，其阻断和破坏雌激素受体。氟维司群是 作为他莫昔芬后疾病进展后的二线治疗引入临床。内分泌治疗是 雌激素受体阳性乳腺癌患者的一线治疗。然而，30%-50%的乳房 经过五至十年治疗的癌症患者获得对内分泌治疗的抵抗力。抗 内分泌治疗和复发是乳腺癌患者死亡的主要原因。脯氨酰羟基化 (P4H)是一种酶，它将脯氨酸羟化为胶原蛋白和其他蛋白质的4-羟脯氨酸， Gly序列作为内质网中的胶原蛋白。P4 H1的α亚基P4 HA 1负责 用于肽结合和催化活性。在这项研究中，我们发现P4 HA 1的表达增加是由于 与内分泌治疗敏感的乳腺癌细胞相比， 细胞P4 HA 1高表达与管腔型乳腺癌的短期无复发生存期显著相关 患者P4 HA 1蛋白致敏的内分泌耐药管腔癌细胞对4-甲基-β-D-半乳糖苷的失活 羟基他莫昔芬（4 OHT）或氟维司群治疗，伴随活性氧（ROS）增加 和内质网（ER）应激。这些初步数据表明，P4 HA 1是一个重要的调节因子， 乳腺癌的内分泌抵抗。我们假设抑制P4 HA 1使管腔型乳腺癌敏感 通过诱导ROS-ER应激通路，将细胞用于内分泌治疗。为了研究P4 HA 1对 内分泌抵抗，我们将介绍P4 HA 1蛋白在内分泌敏感的管腔型乳腺癌中的表达， 细胞系我们将检查对4 OHT或氟维司群治疗的抗性、ER应激水平以及ROS水平。 在4 OHT或氟维司群处理的P4 HA 1过表达细胞中。我们将进行救援实验， 使用ROS抑制剂沉默P4 HA内分泌抗性细胞。对于体内研究，我们将使用内分泌抗性的 管腔型乳腺癌细胞系正交异性异种移植模型，和抗性管腔型乳腺癌患者- 衍生的异种移植物（PDX）模型。我们将确定P4 HA 1的失活是否使这些内分泌 体内对他莫昔芬或氟维司群治疗敏感的耐药肿瘤，并检查是否被淹没 ER应激和ROS水平与肿瘤的内分泌敏感性相关。总之，这次调查将 揭示了P4 HA 1通过调节ROS-ER应激途径在内分泌抵抗中的新的非经典作用， 探索P4 HA 1作为内分泌抵抗型腔型乳腺癌联合治疗的新靶点 癌症患者。

Abstract: Tamoxifen is the oldest and the most widely used selective estrogen receptor modulators. Fulvestrant is a selective estrogen receptor down-regulator which blocks and damages estrogen receptors. Fulvestrant is introduced to clinical as second-line therapy post disease progression after tamoxifen. Endocrine treatment is the first-line therapy for estrogen receptor positive breast cancer patients. However, 30%-50% of the breast cancer patients with five to ten years of treatment acquire resistance to endocrine therapy. Resistance to endocrine therapy and recurrence is the leading cause of mortality in breast cancer patients. Prolyl hydroxylation (P4H), is an enzyme hydroxylates proline into 4-hydroxyproline of collagens and other proteins with the -X-Pro- Gly- sequences as collagens in the endoplasmic reticulum. P4HA1, the alpha subunit of P4H1, is responsible for both peptide binding and catalytic activity. In this study, we found that increased expression of P4HA1 was detected in endocrine therapy resistant luminal breast cancer cells compared with endocrine therapy sensitive cells. High P4HA1 expression significantly correlates with short relapse-free survival in luminal breast cancer patients. Inactivation of P4HA1 protein sensitized endocrine resistance luminal cancer cells to 4- Hydroxytamoxifen (4OHT) or fulvestrant treatment, accompanied by increased reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. These preliminary data indicate that P4HA1 is an important regulator for endocrine resistance of breast cancer. We hypothesize that inhibition of P4HA1 sensitizes luminal breast cancer cells to endocrine therapy by inducing ROS-ER stress pathway. To investigate the importance of P4HA1 for endocrine resistance, we will introduce P4HA1 protein expression in endocrine sensitive luminal breast cancer cell lines. We will examine resistance to 4OHT or fulvestrant treatment, ER stress level, as well as ROS levels in P4HA1-overexpression cells under 4OHT or fulvestrant treatment. We will perform rescue experiments in P4HA-silenced endocrine resistant cells using ROS inhibitor. For the in vivo study, we will use endocrine resistant luminal breast cancer cell lines orthotropic xenograft model, and resistance luminal breast cancer patient- derived-xenograft (PDX) models. We will determine whether inactivation of P4HA1 renders these endocrine resistant tumors sensitive to tamoxifen or fulvestrant treatment in vivo, and examine whether the overwhelmed ER stress and ROS levels are associated with endocrine sensitivity in tumors. In summary, this investigation will uncover a novel uncanonical role of P4HA1 in endocrine resistance by regulation ROS-ER stress pathway, and explore P4HA1 as a novel therapeutic target for combinatorial therapy for endocrine resistant luminal breast cancer patients.