Singh Chandra K, George Jasmine, Chhabra Gagan, Nihal Minakshi, Chang Hao, Ahmad Nihal
Department of Dermatology, University of Wisconsin, Madison, WI, United States.
William S. Middleton VA Medical Center, Madison, WI, United States.
Front Oncol. 2021 Apr 16;11:676077. doi: 10.3389/fonc.2021.676077. eCollection 2021.
The mitochondrial sirtuin SIRT3 plays key roles in cellular metabolism and energy production, which makes it an obvious target for the management of cancer, including melanoma. Previously, we have demonstrated that SIRT3 was constitutively upregulated in human melanoma and its inhibition resulted in anti-proliferative effects in human melanoma cells and in human melanoma xenografts. In this study, we expanded our data employing knockdown and overexpression strategies in cell culture and mouse xenografts to further validate and establish the pro-proliferative function of SIRT3 in melanocytic cells, and its associated potential mechanisms, especially focusing on the metabolic regulation. We found that short-hairpin RNA (shRNA) mediated SIRT3 knockdown in G361 melanoma cells showed diminished tumorigenesis in immunodeficient Nu/Nu mice. Conversely, SIRT3 overexpressing Hs294T melanoma cells showed increased tumor growth. These effects were consistent with changes in markers of proliferation (PCNA), survival (Survivin) and angiogenesis (VEGF) in xenografted tissues. Further, in culture system, we determined the effect of SIRT3 knockdown on glucose metabolism in SK-MEL-2 cells, using a PCR array. SIRT3 knockdown caused alterations in a total of 37 genes involved in the regulation and enzymatic pathways of glucose (32 genes) and glycogen (5 genes) metabolism. Functions annotation of these identified genes, using the ingenuity pathway analysis (IPA), predicted cumulative actions of decreased cell viability/proliferation, tumor growth and reactive oxygen species (ROS), and increased apoptosis in response to SIRT3 knockdown. Further, IPA gene network analysis of SIRT3 modulated genes revealed the interactions among these genes in addition to several melanoma-associated genes. Sirtuin pathway was identified as one of the top canonical pathways showing the interaction of SIRT3 with metabolic regulatory genes along with other sirtuins. IPA analysis also predicted the inhibition of HIF1α, PKM, KDM8, PPARGC1A, mTOR, and activation of P53 and CLPP; the genes involved in major cancer/melanoma-associated signaling events. Collectively, these results suggest that SIRT3 inhibition affects cellular metabolism, to impart an anti-proliferative response against melanoma.
线粒体去乙酰化酶SIRT3在细胞代谢和能量产生中发挥关键作用，这使其成为包括黑色素瘤在内的癌症治疗的一个明显靶点。此前，我们已经证明SIRT3在人类黑色素瘤中组成性上调，其抑制导致人类黑色素瘤细胞和人类黑色素瘤异种移植瘤产生抗增殖作用。在本研究中，我们在细胞培养和小鼠异种移植瘤中采用敲低和过表达策略扩展了我们的数据，以进一步验证和确立SIRT3在黑素细胞中的促增殖功能及其相关潜在机制，尤其关注代谢调节。我们发现，短发夹RNA（shRNA）介导的G361黑色素瘤细胞中SIRT3敲低在免疫缺陷的裸鼠中显示肿瘤发生减少。相反，过表达SIRT3的Hs294T黑色素瘤细胞显示肿瘤生长增加。这些效应与异种移植组织中增殖标志物（增殖细胞核抗原）、存活标志物（生存素）和血管生成标志物（血管内皮生长因子）的变化一致。此外，在培养系统中，我们使用PCR阵列确定了SIRT3敲低对SK-MEL-2细胞葡萄糖代谢的影响。SIRT3敲低导致总共37个参与葡萄糖（32个基因）和糖原（5个基因）代谢调节及酶促途径的基因发生改变。使用 Ingenuity 通路分析（IPA）对这些已鉴定基因进行功能注释，预测了细胞活力/增殖降低、肿瘤生长和活性氧（ROS）减少以及响应SIRT3敲低凋亡增加的累积作用。此外，对SIRT3调节基因的IPA基因网络分析揭示了这些基因之间以及几个黑色素瘤相关基因之间的相互作用。去乙酰化酶通路被确定为顶级经典通路之一，显示SIRT3与代谢调节基因以及其他去乙酰化酶之间的相互作用。IPA分析还预测了缺氧诱导因子1α、丙酮酸激酶M2、赖氨酸特异性去甲基化酶8、过氧化物酶体增殖物激活受体γ共激活因子1α、哺乳动物雷帕霉素靶蛋白的抑制以及P53和ClpP的激活；这些基因参与主要的癌症/黑色素瘤相关信号事件。总体而言，这些结果表明SIRT3抑制影响细胞代谢，从而对黑色素瘤产生抗增殖反应。
