Sherapura Ankith, Kiran B K, Pavan Kumar G S, Siddesh B M, Thirusangu Prabhu, Suchetha Kumari N, Prabhakar B T
Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga, 577203, Karnataka, India.
Department of Experimental Pathology and Laboratory Medicine, Mayo Clinic, Rochester, MN, USA.
Chem Biol Interact. 2025 Jan 25;406:111319. doi: 10.1016/j.cbi.2024.111319. Epub 2024 Nov 27.
Metastasis is complex and insidious type of disease involves multiple signaling nexus, which have implications in understanding disease pathogenesis. Treatment failure for metastatic cancer is frequently high due to aggressive adaptation of cancerous cells to invade to neighboring organs. Cytoskeleton intermediate filamentous protein Vimentin and scaffolding protein Neural precursor cell expressed Developmentally Down-regulated protein 9 (NEDD9) play a key role in metastatic events by regulating multiple metastatic events. Interaction between these proteins is necessary to promote metastatic progression. Withaferin A (WFA), a natural pharamacophore, known to target Vimentin to induce antitumor potential. However exact molecular mechanism still yet to be elucidated. We hypothesize, Vimentin-NEDD9 signaling nexus is necessary for metastatic progression and targeting this interwoven signaling loop with effective pharamacophore WFA halts metastatic progression of melanoma into lung. To elucidate the same, we carried out gene expression measurement through quantitative Reverses Transcription Polymerase Chain Reaction (qRT-PCR), Immunoblot and Immunohistochemistry. Assessment of interactive signaling by Co-immunoprecipitation, Immunofluorescence, Co-localization and Proximity ligation assay. Phosphorylation studies through transfection of phospho specific mutant constructs generated through site directed mutagenesis. WFA induced cellular behavioral changes by migration, invasion assays and Immunoblot analysis. The B16F10 induced mouse metastatic melanoma model to asses NEDD9-Vimentin expression and anti-metastasis induced by WFA. The results postulates, elevated levels and interaction between NEDD9-Vimentin proteins, have positive correlation in metastatic progression of melanoma into lung in both in-vitro and in-vivo condition, establishing it as therapeutic target. Pharmacologically, WFA targets this complex by extending its activity by not only inducing specific Serine 56 phosphorylation of Vimentin, also dissociates NEDD9 signaling loop to halt Epithelial-mesenchymal transition (EMT) and subsequent metastatic events. Eventually, modulation of the relevant metastatic genes E-Cadherin, N-Cadherin, SNAIL, MMP-2 & MMP-9 resulted in regression of metastatic melanoma progression to lung. The study validates WFA induced S56 phosphorylation is necessary to abrupt the NEDD9-Vimentin metastatic signaling complex to regress aggressive metastatic melanoma. The investigation emphasized more mechanistic approach of WFA. Understanding and targeting such integrative mechanical input in the tumor microenvironment will be a better therapeutic strategy to combat metastasis.
转移是一种复杂且隐匿的疾病类型，涉及多个信号网络，这对于理解疾病发病机制具有重要意义。由于癌细胞具有侵袭邻近器官的强烈适应性，转移性癌症的治疗失败率通常较高。细胞骨架中间丝蛋白波形蛋白和支架蛋白神经前体细胞表达的发育下调蛋白9（NEDD9）通过调节多个转移事件在转移过程中起关键作用。这些蛋白质之间的相互作用对于促进转移进展是必要的。Withaferin A（WFA）是一种天然药效基团，已知可靶向波形蛋白以诱导抗肿瘤潜力。然而，确切的分子机制仍有待阐明。我们假设，波形蛋白-NEDD9信号网络对于转移进展是必要的，并且用有效的药效基团WFA靶向这个交织的信号环可阻止黑色素瘤向肺部的转移进展。为了阐明这一点，我们通过定量逆转录聚合酶链反应（qRT-PCR）、免疫印迹和免疫组织化学进行基因表达测量。通过免疫共沉淀、免疫荧光、共定位和邻近连接分析评估相互作用信号。通过转染通过定点诱变产生的磷酸化特异性突变构建体进行磷酸化研究。WFA通过迁移、侵袭试验和免疫印迹分析诱导细胞行为变化。利用B16F10诱导的小鼠转移性黑色素瘤模型来评估NEDD9-波形蛋白的表达以及WFA诱导的抗转移作用。结果表明，NEDD9-波形蛋白的水平升高及相互作用在体外和体内条件下黑色素瘤向肺部的转移进展中均呈正相关，将其确立为治疗靶点。在药理学上，WFA通过扩展其活性来靶向这个复合物，不仅诱导波形蛋白特定的丝氨酸56磷酸化，还解离NEDD9信号环以阻止上皮-间质转化（EMT）及随后的转移事件。最终，对相关转移基因E-钙黏蛋白、N-钙黏蛋白、蜗牛蛋白、基质金属蛋白酶-2和基质金属蛋白酶-9的调节导致转移性黑色素瘤向肺部进展的消退。该研究证实WFA诱导的S56磷酸化对于破坏NEDD9-波形蛋白转移信号复合物以消退侵袭性转移性黑色素瘤是必要的。该研究强调了WFA更多的作用机制。理解并靶向肿瘤微环境中的这种综合机械输入将成为对抗转移的更好治疗策略。
