成骨细胞壁龛中PKD1调节的成骨细胞分化对骨髓播散前列腺癌细胞休眠作用及机制

The colonization and dormancy of disseminated tumor cells (DTCs) on the osteoblastic niche in the bone marrow microenvironment represent a major characteristic of various bone metastatic tumors including prostate cancer, and are responsible for the chemotherapeutic resistance. Our previous results and preliminary data showed that conditional knockout of PKD1 in pre-osteoblastic lineage suppresses osteoblast differentiation and osteopontin(OPN)secretion, leading to the prostate cancer cells growth in vitro co-culture system. By the contrast, adding of OPN to the co-cultural medium results in inhibition of tumor cells growth. We hypothesize for the first time that PKD1 may contribute to the DTCs colonization and dormancy on the osteoblastic niche through regulation of osteoblast differentiation and OPN secretion in the bone marrow microenvironment. To test this hypothesis, the proposed study will utilize the following AIMs: AIM 1 :Verify the osteoblast differentiation and OPN secretion induced by PKD1 in co-culture contributes to the prostate cancer cells growth ; AIM 2: Confirm the inhibition of osteoblast differentiation and OPN secretion triggered by conditional knockout of PKD1 in preosteoblastic lineage of mice could release from tumor cells colonization and dormancy on the osteoblastic niche, leading to the sensitivity to the chemotherapy using conditional knockout mice and bone metastasis mice model; AIM 3: Explore the mechanism of PKD1 contributions to DTCs colonization and dormany through regulation of osteoblast differentiation in the bone marrow microenvironment. This proposal will provide new insights into the intrinsic correlation of osteoblast differentiation and osteoblastic niche with prostate cancer DTCs colonization and dormancy in the bone marrow microenvironment.

包括前列腺癌在内的多种肿瘤在出现明显的骨转移前，骨髓内播散肿瘤细胞（DTCs）常在成骨细胞壁龛上定居及休眠，因而产生化疗药物治疗性抵抗。前期研究及预实验发现，敲除前体成骨细胞PKD1则抑制成骨细胞分化及骨桥蛋白(OPN)的分泌，并促进共培养的前列腺癌细胞生长；相反，添加OPN则抑制共培养的前列腺癌细胞生长。据此提出假设：PKD1可能通过调节成骨细胞分化及OPN分泌促进骨髓微环境中DTCs在成骨细胞壁龛粘附、定居及休眠，最终产生化疗抵抗。为此，拟探讨：（1）细胞共培养证实PKD1调节的成骨细胞分化及OPN的分泌促进前列腺癌细胞休眠；（2）体内肿瘤骨转移模型确证PKD1敲除鼠中抑制成骨细胞分化及OPN的分泌解除DTCs的定居和休眠，并增强化疗敏感性；（3）探讨PKD1调节的成骨细胞分化及OPN的分泌促进DTCs休眠的分子机制。本研究为揭示成骨细胞分化与DTCs定居及休眠的内在关联提供新的视野。

近年的研究表明，PKD1参与高尔基体反面膜转运、成骨细胞的分化、细胞存活、衰老、血管新生等多种细胞功能调节。但是，PKD1是否通过调节成骨细胞的分化进而促进播散肿瘤细胞在骨髓中的休眠尚不清楚。本项目通过一系列细胞、分子生物学实验证实成骨细胞中PKD1通过调节CREB1的表达和活性来促进GAS6的表达和分泌，进而调节前列腺癌细胞的休眠。此外，成骨细胞中PKD1的表达通过上调GAS6的分泌显著促进前列腺癌细胞PC-3中节律相关基因PER1、PER2、PER3、CLOCK、BMAL1和DEC2的表达，最终促进前列腺癌细胞的休眠。结果显示：（1）成骨细胞中过表达PKD1促进前列腺癌细胞的休眠,表现为前列腺癌细胞表面DiD的代谢速率减缓、KI-67的表达降低以及ERK/p38活性比的下降。（2）敲低成骨细胞中的内源性PKD1抑制前列腺癌细胞的休眠，表现为前列腺癌细胞表面DiD的代谢加快、KI-67的表达增加以及ERK/p38 活性比值回升。（3）成骨细胞中上调PKD1的表达促进GAS6的表达和分泌。相反的，敲低PKD1则抑制GAS6的表达和分泌。（4）成骨细胞中PKD1与CREB1存在相互作用，PKD1促进CREB1的表达和磷酸化。（5）成骨细胞中PKD1 的过表达促进了 CREB1 与 Gas6 启动子区域结合的能力。（6）GAS6促进前列腺癌细胞中节律相关基因CLOCK、BMAL1、PER1、PER2和DEC2的表达。（7）前列腺癌的临床TCGA 数据分析显示，AXL和MERTK 的表达与生物钟基因PER1、PER2、PER3、CLOCK、BMAL1和DEC2的表达相关，且CLOCK、BMAL1和DEC2的表达与前列腺癌的复发相关。上述研究为进一步揭示前列腺癌在骨髓中休眠的分子机制奠定基础，并为临床治疗前列腺癌的骨转移提供新的思路。