在將近50%左右的黑色素瘤病患當中都有發現致癌基因BRAF的突變，其中又有高達90%以上的突變都發生在第600個胺基酸位點，由纈胺酸（Valine）突變成麩胺酸(Glutamic acid)。臨床上針對這些有BRAFV600E突變的黑色素瘤患者可以使用BRAF抑制劑，但是當患者持續用藥大約6-12個月之後就會產生抗藥性
HSP90抑制劑的功效在許多癌症當中進行測試，並且已經進入臨床測試階段。第二代的HSP90抑制劑有抗腫瘤生長的功能，但在黑色素瘤仍然不明瞭，因此我們想要測試AUY922對於抑制黑色素癌的能力，進而找尋新的HSP90受質，探討此受質與黑色素癌的生長與抗藥性的產生是否有關。
本研究發現TRPM1為新穎HSP90的受質，AUY922能破壞TRPM1和HSP90之間的交互作用。我們也發現黑色素瘤細胞株確實有TRPM1 蛋白表現，而我們建立的BRAF抑制劑抗性的黑色素瘤細胞株(DR)會有比較高表現的。在TRPM1敲除之後會抑制黑色素瘤細胞株活性以及形成細胞群體的能力，透過抑制AKT訊號活化，增加caspase 3的活性，最後導致細胞凋亡。AUY922能降低TRPM1蛋白表現，亦會抑制黑色素瘤細胞株生長，使AKT失活而誘導細胞凋亡。此現象亦發生在BRAF抑制劑抗性的黑色素瘤細胞。我們進一步解開了AUY922調降TRPM1蛋白表現的分子機轉，發現AUY922不影響HSP90-HSP70、HSP70-TRPM1的交互作用, 但是AUY922破壞CDC37與HSP90的交互作用。我們也證實CDC37 shRNA的表達會調降TRPM1蛋白表現，由此可知CDC37調控了HSP90對於TRPM1的穩定。最後，由人類皮下腫瘤小鼠模式中證實，AUY922能降低腫瘤生長速度，並且能再次抑制Dabrafenib抗藥性腫瘤生長。
綜上所述，TRPM1是新穎HSP90的受質蛋白，對黑色素瘤生長非常重要。AUY922會破壞CDC37與HSP90複合物的交互作用, 而降低TRPM1蛋白, 促使細胞凋亡，抑制腫瘤生長。BRAF抑制劑和AUY922的合併療法仍須要進一步的臨床前測試，有機會能增進對黑色素瘤的控制。

The mutations of BRAF are found in approximately half of the melanoma patients. The BRAFV600E mutation is identified up to 90% of the BRAF mutations. The BRAF inhibitors have been shown high response rates and improve survival in melanoma patients with BRAFV600E mutation, but a majority of these patients develop drug resistance within 6-12 months. Thus, we try to find out the other drugs or protein targets for treating aggressive melanoma patients.
The HSP90 inhibitors (HSP90i) have under clinical evaluating for the treatments in different cancer types. AUY922, an HSP90i, has been tested in lung cancers, gastric cancers, breast cancers, and leukemia with modest anti-tumor activity. The anti-tumor activity of AUY922 hasn’t been demonstrated in melanoma yet, so we wonder the performance of AUY922 in melanoma. Moreover, we hope to identify a new HSP90 client protein which is responsible for the efficacy of AUY922 and overcome the resistance of BRAFi in melanoma.
We characterized that TRPM1 is a novel HSP90 client protein by AUY922 disrupted the interaction between HSP90 protein complex and TRPM1. The interactions between HSP90-HSP70 and HSP70-TRPM1 were not affected by AUY922 treatment. However, CDC37 was disassociated with HSP90 before the degradation of TRPM1 in response to AUY922. In addition, the TRPM1 protein was declined in CDC37-knockdown cells which suggested that CDC37 is important for the stability of TRPM1.
We found that TRPM1 was expressed in malignant melanoma cells and the Dabrafenib-resistance (DR) melanoma cell lines had a higher expression level of TRPM1. The knockdown of TRPM1 inhibited the cell viability and growth of melanoma cells by reducing the AKT activation. Therefore, caspase 3 was activated and promoted cell apoptosis. Similarly, AUY922 induced the degradation of TRPM1, inactivation of AKT, and cell apoptosis. Finally, the A375 xenograft animal models showed that AUY922 suppressed the growth of melanoma tumor and Dabrafenib-resistant tumor.
In summary, TRPM1 is a novel HSP90 client protein. TRPM1 is essential for the survival of melanoma. AUY922 decreases the TRPM1 expression, inhibits AKT signaling, promotes the cell apoptosis, and suppresses xenograft tumor growth. The anti-tumor effects of AUY922 on Dabrafenib-resistant tumor is an important finding that provides a rationale for evaluating the combination treatment of BRAFi and AUY922 in melanoma patients.

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