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不治之症。 转移瘤。破骨细胞激活和成骨细胞抑制导致骨丢失、病理性骨折、 高钙血症和骨痛，常伴有骨骼肌丧失(恶病质)。当前的治疗目标 破骨细胞和对肿瘤生长的影响有限，同时不能修复丢失的骨骼或肌肉。 肿瘤分泌的甲状旁腺激素相关蛋白，PTHrP，是骨破坏的主要原因，通过 诱导RANKL表达，增加破骨细胞数量和功能。转化生长因子β，在骨骼中释放 吸收，刺激乳腺癌产生甲状旁腺素rP[Yen等人，1999]，驱动骨骼的恶性循环 转移[Chirgwin&Guise，2000；Kakonen等人，2002]。PTHrP刺激PTH1R受体生长- 表达肿瘤并通过脂肪细胞的重新编程间接导致癌症恶病质[Kir&Spiegelman， 2016年]。可引起恶性肿瘤的高钙血症，刺激血管生成和癌性骨痛。我们相信 肿瘤PTHrP的有害作用可被独特的PTH1R配体阻断，同时具有有益的合成代谢 对骨骼的影响--这是PTHrP中和抗体所不能达到的。[D-Trp12，Tyr34]-牛-PTH(7- 34)，bPTH7-34DD是一种独特的通过PTH1R降低cAMP的G蛋白偶联信号的反向激动剂，而 激活β-arrestin信号。它促进骨形成而不会引起高钙血症或骨溶解。 [Maudsley等人，2015年]。我们的数据显示了bPTH7-34DD的抗骨溶解和抗肿瘤作用。 伴有鼠骨的乳腺癌。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bPTH7-34DD将：1)抑制溶骨性骨转移的恶性循环 骨溶解和肿瘤生长；2)通过阻止脂肪细胞从白色向棕色转化来减少癌症恶病质 骨；3)通过刺激新骨形成来保持骨健康；4)有效治疗溶骨性肿瘤 乳腺癌。5)对其他类型的PTHrP分泌的肿瘤有效。BPTH7-34DD将在异种移植中进行测试 乳腺癌骨转移模型，在预防和治疗环境中。BPTH7-34DD的作用机制 信号将在癌细胞与小鼠骨骼以及骨和脂肪细胞系的体外共培养中进行测试，并 10个细胞，取自wt&β-arrestin k/o(βarr2-/-)小鼠骨和细胞。 具体目的1)测定bPTH7-34DD对肿瘤细胞的体外生长效应；b)确定其作用机制 对成骨细胞(Obs)、破骨细胞(OCs)和骨细胞(Ots)的作用；ii)对脂肪细胞的作用；c)测试是否有反应 BPTH7-34DD需要β-arrestin，使用βarr2-/-小鼠的骨和细胞；d)测试bPTH7-34DD是否阻碍生长 前列腺癌、肺癌和多发性骨髓瘤细胞的体外骨溶解和骨溶解 具体目标2：在体内测试bPTH7-34DD对以下方面的显著影响：a)肿瘤负担；b)溶骨性损害；c) 全身和邻近肿瘤的骨丢失/形成；d)骨骼肌。 意义：bPTH7-34DD可迅速进入癌症骨转移的临床试验。相似的PTH &PTHrP多肽用于治疗骨质疏松症。益处可能延伸到其他表达PTHrP的癌症 转移到骨，包括多发性骨髓瘤，这也定植于骨并导致骨溶解。 技术方法：该项目使用癌细胞与小鼠骨骼共培养，称为Evoca(体外活体 器官共培养试验)。利用稳定转导的分泌型赤子荧光素酶(GLuc)检测肿瘤生长 癌细胞。骨骼和MM基因的变化通过老鼠和人类特异的qPCR进行量化。两只鼠标 乳腺癌骨转移模型将用于临床前测试。顾问：Louis Luttrell博士是 世界甲状旁腺素信号专家，并破译了bPTH7-34DD的作用。特蕾莎·齐默斯博士是一位专家 癌症恶病质。Jesse 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方面的世界专家。

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.