The role of MAPK 38 in resistance of breast cancer and osteolytic bone metastases to inhibitors of the mevalonate pathway

甲羟戊酸途径是重要的信号通路，可被双膦酸盐和他汀类药物阻断。当双膦酸盐抑制骨吸收破骨细胞时，他汀类药物被用来降低胆固醇的合成。大量研究描述了这两种药物的直接和间接抗肿瘤作用。然而，高浓度，可能超过临床实践中可达到的水平，需要获得显著的效果。在第一个资助期，我们证明了使用他汀类药物和唑来膦酸序贯阻断甲羟戊酸途径可显著降低这些药物在人类乳腺癌细胞中达到有效抗肿瘤作用所需的浓度。此外，使用该方法观察到DKK-1的显著抑制。DKK-1是成骨细胞分化的抑制因子，并已成为骨溶解性骨转移的关键调节因子。然而，他汀类药物和唑来膦酸治疗激活了丝裂原活化蛋白激酶（MAPK） p38，药物抑制p38增强了阿托伐他汀对乳腺癌细胞凋亡的诱导。此外，p38在人类癌细胞中直接调控DKK-1。基于这些发现，我们假设p38的激活导致乳腺癌细胞对甲羟戊酸途径抑制剂的抗肿瘤活性产生耐药性。因此，我们认为阻断p38是使人乳腺癌细胞对他汀类药物和唑来膦酸的抗肿瘤和DKK-1抑制作用敏感的一种有希望的方法。为了验证这一假设，我们首先旨在全面表征同时抑制p38和甲羟戊酸途径介导的抗肿瘤作用。具体来说，我们打算确定p38的各自同种异构体，这些异构体负责观察到的抗性。我们的发现将通过使用合适的动物模型来证实原发肿瘤生长和骨溶解性骨转移。在这里，将在肿瘤细胞中使用RNA干扰或药物抑制剂敲除p38，并在肿瘤细胞接种后用他汀类药物和唑来膦酸治疗小鼠。因此，我们将评估肿瘤细胞中DKK-1表达的降低是否以及如何可能潜在地减少骨转移中溶骨性病变的发生。为了强调我们研究结果的重要性，我们将分析原发性乳腺癌组织，以确定甲羟戊酸途径活性与DKK-1和p38表达之间的联系。我们的研究旨在确定乳腺癌细胞对甲羟戊酸途径抑制剂的重要耐药机制，该机制可能导致临床剂量的他汀类药物和唑来膦酸的抗肿瘤效果无效。

The mevalonate pathway is an essential signaling pathway that can be blocked by bisphosphonates and statins. While bisphosphonates inhibit bone-resorbing osteoclasts, statins are used to lower the synthesis of cholesterol. Numerous studies describe direct and indirect anti-tumor effects by both drugs. However, high concentrations, which likely exceed those attainable in clinical practice, are required to obtain significant effects. In the first funding period, we demonstrated that sequential blockade of the mevalonate pathway using statins and zoledronic acid significantly lowers the concentrations of these drugs needed to achieve effective anti-tumor effects in human breast cancer cells. In addition, a significant suppression of DKK-1 was observed using this approach. DKK-1 is an inhibitor of osteoblastic differentiation and has emerged as a key modulator of osteolytic bone metastases. However, the treatment with statins and zoledronic acid activated the mitogen activated protein kinase (MAPK) p38, and pharmacological inhibition of p38 enhanced the induction of apoptosis by atorvastatin in breast cancer cells. Furthermore, a direct regulation of DKK-1 by p38 was demonstrated in human cancer cells.Based on these findings we hypothesize that activation of p38 results in a breast cancer cell resistance to the anti-tumor activities of mevalonate pathway inhibitors. Thus, we consider the blockade of p38 as a promising approach to sensitize human breast cancer cells to the anti-tumor and DKK-1 suppressive effects of statins and zoledronic acid. To test this hypothesis, we first aim to comprehensively characterize the anti-tumor effects mediated by the simultaneous inhibition of p38 and the mevalonate pathway. Specifically, we intend to identify the respective isoforms of p38 that are responsible for the observed resistance. Our findings will be confirmed by using appropriate animal models of both primary tumor growth and osteolytic bone metastases. Here, p38 will be knocked down using RNA interference or pharmacological inhibitors in the tumor cells, and mice will be treated with statins and zoledronic acid after tumor cell inoculation. Thus, we will assess if and how the reduced expression of DKK-1 in the tumor cells might potentially reduce the occurrence of osteolytic lesions in bone metastases. To underline the significance of our findings, primary breast cancer tissue will be analyzed to determine a link between the mevalonate pathway activity with the expression of DKK-1 and p38. Our study aims to identify an important resistance mechanism of breast cancer cells against inhibitors of the mevalonate pathway that might contribute to ineffective anti-tumor effects observed with clinical doses of statins and zoledronic acid.