A Novel Agent to Suppress Tumor Growth in Bone, Prevent Cachectic Muscle Loss and Preserve Skeletal Integrity

摘要：超过85%的晚期乳腺癌和前列腺癌患者患有无法治愈的骨癌 转移破骨细胞活化和成骨细胞抑制导致骨丢失、病理性骨折， 高钙血症和骨痛，通常伴有骨骼肌损失（恶病质）。当前治疗目标 破骨细胞，并且对肿瘤生长的作用有限，同时不能恢复丢失的骨或肌肉。 肿瘤分泌的甲状旁腺相关蛋白PTHrP是骨破坏的主要原因， 诱导RANKL表达以增加破骨细胞数量和功能。TGFβ，骨形成过程中释放 吸收，刺激乳腺癌产生PTHrP [Yin et al，1999]，驱动骨的恶性循环 转移[Chirgwin & Guise，2000; Kakonen et al，2002]。PTHrP刺激PTH 1 R受体的生长- 表达肿瘤并通过脂肪细胞的重编程间接引起癌症恶病质[Kir & Spiegelman， 2016年]。它引起恶性肿瘤的高钙血症，刺激血管生成和癌症骨痛。我们相信 肿瘤PTHrP的有害作用可以被独特的PTH 1 R配体阻断，同时具有有益的合成代谢， 对骨骼的影响-这是PTHrP中和抗体无法实现的。[D-Trp 12，Tyr 34]-牛-PTH（7- 34），bPTH 7 - 34 DD，是通过PTH 1 R的G蛋白偶联信号传导的独特反向激动剂，降低cAMP，而 激活β-抑制蛋白信号传导。它增加骨形成而不引起高钙血症或骨质溶解 [Maudsley等人，2015]。我们的数据显示bPTH 7 - 34 DD在与人成骨细胞共培养物中的抗溶骨性和抗肿瘤作用。 乳腺癌的老鼠骨头。它还对抗与癌症恶病质相关的脂肪细胞基因变化。 我们假设bPTH 7 - 34 DD将：1）通过抑制骨转移瘤的恶性循环，抑制溶骨性骨转移瘤的恶性循环。 骨质溶解和肿瘤生长; 2）通过阻断白细胞向棕色脂肪细胞的转化减少癌症恶病质， 骨; 3）通过刺激新骨形成来保持骨健康; 4）有效治疗由于骨溶解性肿瘤而导致的骨损伤。 乳腺癌5)对其他PTHrP分泌型肿瘤有效。将在异种移植物中检测bPTH 7 - 34 DD 乳腺癌骨转移模型，在预防和治疗设置。bPTH 7 - 34 DD的作用机制 将在癌细胞与小鼠骨的离体共培养物中以及在骨和脂肪细胞系中测试信号传导， 10细胞以及来自wt & β-arrestin k/o（β arr 2-/-）小鼠的骨和细胞。 具体目的1：a）确定bPTH 7 - 34 DD对肿瘤细胞的离体生长作用; B）确定其机制 对i）成骨细胞（Obs）、破骨细胞（Ocs）和骨细胞（Ots）的作用; ii）对脂肪细胞的作用; c）测试是否响应 使用来自β arr 2-/-小鼠的骨和细胞，测试bPTH 7 - 34 DD是否阻断生长 在前列腺癌、肺癌和多发性骨髓瘤细胞离体骨和骨质溶解中 具体目标2：体内测试bPTH 7 - 34 DD对以下各项的显著影响：a）肿瘤负荷; B）溶骨性病变; c） 全身性和邻近肿瘤的骨丢失/形成; d）骨骼肌。 意义：bPTH 7 - 34 DD可以快速进入癌症骨转移的临床试验。相似PTH & PTHrP肽用于治疗骨质疏松症。受益可能扩展到其他PTHrP表达癌症， 转移到骨，包括多发性骨髓瘤，其也定植于骨并引起骨质溶解。 技术方法：该项目使用癌细胞与小鼠骨骼的共培养物，称为EVOCA（离体 器官共培养测定）。通过来自稳定转导的细胞的分泌的Gaussia荧光素酶（gLuc）测定肿瘤生长。 癌细胞通过小鼠和人特异性qPCR定量骨和MM基因的变化。两个鼠标 乳腺癌骨转移模型将用于临床前试验。顾问：Louis Luttrell博士是 他是PTH信号传导方面的世界级专家，并破译了bPTH 7 - 34 DD的作用。特蕾莎·齐默斯医生是一位 在癌症恶病质中。Jesus Delgado-Calle博士是骨细胞生物学和骨细胞：脂肪细胞方面的专家 骨髓瘤中的相互作用。大卫鲁德曼博士是骨髓瘤骨病的世界专家。

ABSTRACT: Over 85% of patients with advanced cancers of breast and prostate suffer from incurable bone metastases. Osteoclast activation and osteoblast suppression lead to bone loss, pathologic fractures, hypercalcemia and bone pain, often accompanied by loss of skeletal muscle (cachexia). Current therapies target osteoclasts and have limited effects on tumor growth, while not restoring lost bone or muscle. Tumor-secreted parathyroid hormone-related protein, PTHrP, is a major causal agent of bone destruction by inducing expression of RANKL to increase osteoclast number and function. TGFβ, released during bone resorption, stimulates breast cancer production of PTHrP [Yin et al, 1999], driving a vicious cycle of bone metastases [Chirgwin & Guise, 2000; Kakonen et al, 2002]. PTHrP stimulates growth of PTH1R receptor- expressing tumors and indirectly causes cancer cachexia via reprogramming of adipocytes [Kir & Spiegelman, 2016]. It causes hypercalcemia of malignancy, stimulates angiogenesis and cancer bone pain. We believe the deleterious actions of tumor PTHrP can be blocked by a unique PTH1R ligand, while having beneficial anabolic effects on bone - which cannot be achieved with PTHrP-neutralizing antibody. [D-Trp12,Tyr34]-bovine-PTH(7- 34), bPTH7-34DD, is a unique inverse agonist of G protein-coupled signaling by PTH1R, decreasing cAMP, while activating β-arrestin signaling. It increases bone formation without causing hypercalcemia or osteolysis [Maudsley et al, 2015]. Our data showed anti-osteolytic and anti-tumor actions of bPTH7-34DD in co-cultures of breast cancer with mouse bone. It also countered adipocyte gene changes associated with cancer cachexia. We hypothesize that bPTH7-34DD will: 1) Inhibit the vicious cycle of osteolytic bone metastases by suppressing osteolysis & tumor growth; 2) Decrease cancer cachexia by blocking white-to-brown adipocyte conversion in bone; 3) Preserve bone health by stimulating new bone formation; 4) Effectively treat osteolytic tumors due breast cancer. 5) Be effective against other PTHrP-secreted tumor types. bPTH7-34DD will be tested in xenograft models of breast cancer bone metastasis, in prevention and treatment settings. The mechanism of bPTH7-34DD signaling will be tested in ex vivo co-cultures of cancer cells with mouse bones and in bone and fat cell lines and 1o cells and with bones and cells from wt & β-arrestin k/o (βarr2-/-) mice. Specific Aim 1: a) Determine growth effects ex vivo of bPTH7-34DD on tumor cells; b) Determine the mechanism of action of on i) Osteoblasts (Obs), osteoclasts (Ocs), & osteocytes (Ots); ii) On adipocytes; c) Test if responses to bPTH7-34DD require β-arrestin, using bones & cells from βarr2-/- mice; d) Test if bPTH7-34DD blocks growth in bone and osteolysis ex vivo with prostate and lung cancer and multiple myeloma cells Specific Aim 2: Test bPTH7-34DD in vivo for significant effects on: a) Tumor burden; b) Osteolytic lesions; c) Bone loss/formation systemically and adjacent to tumor; d) Skeletal muscle. Significance: bPTH7-34DD could be rapidly brought into clinical trials for cancer bone metastasis. Similar PTH & PTHrP peptides are used to treat osteoporosis. Benefit may extend to other PTHrP-expressing cancers that metastasize to bone, including multiple myeloma, which also colonizes bone and causes osteolysis. Technical Approaches: The project uses co-cultures of cancer cells with mouse bones, called EVOCA (ex vivo organ co-culture assay). Tumor growth is assayed by secreted Gaussia luciferase (gLuc) from stably transduced cancer cells. Changes in bone and MM genes are quantified by mouse- and human-specific qPCR. Two mouse models of breast cancer bone metastases will be used for preclinical testing. Consultants: Dr. Louis Luttrell is a world expert in PTH signaling and deciphered the actions of bPTH7-34DD. Dr. Teresa Zimmers is an expert in cancer cachexia. Dr. Jesus Delgado-Calle is an expert in osteocyte biology and osteocyte:adipocyte interactions in myeloma. Dr. David Roodman is a world expert on myeloma bone disease.