Genetic Manipulation of Sirtuin 3 Causes Alterations of Key Metabolic Regulators in Melanoma

Abstract

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 vitro in human melanoma cells and in vivo 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 in vitro 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.

Mitochondrial Metabolism in Melanoma

“…In addition, the experimental overexpression of SIRT3 via plasmids in Hs294T human melanoma cells and immortalized melanocytes led to both enhanced proliferation and colony formation in the melanoma cells and increased proliferation in melanocytes, evidence of the role of SIRT3 in melanoma growth [33]. This result was supported by observations that tumors with an induced overexpression of SIRT3 in mouse xenograft tumors had increased tumorigenicity [153]. Furthermore, short hairpin RNA knockdown of SIRT3 in melanoma led to favorable effects including reduced cell proliferation and migration, indicating possible therapeutic options [33].…”

“…Furthermore, SIRT3 has a general role in maintaining functional metabolism, as the knockdown of SIRT3 led to alterations in genes for multiple biochemical pathways, including glycolysis, the citric acid cycle, and the pentose phosphate pathway [153]. One key function of SIRT3 is reduction in ROS via the detoxification enzyme manganese superoxide dismutase 2 (SOD2).…”

“…Through SIRT3, honokiol derivatives might have driven mitochondrial fusion, a morphology favoring oxidative phosphorylation [154]. Tumors which have high levels of SIRT3 in vivo may have normal or nearly normal mitochondria due to the important regulatory role of SIRT3, while tumors which have lower levels of SIRT3 may have mitochondrial abnormalities and may be susceptible to SIRT3 activation [153]. Furthermore, inducing SIRT3 could reduce MCL1, decreasing resistance to apoptosis, while shifting away from a glycolytic metabolism could decrease chemoresistance mediated by the nuclear factor kappa B complex [18,155].…”

Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype

“…In our analysis we have found inconsistent finding with state of downstream molecule (yellow lines present in Fig 9 ). We would like to emphasize that the inconsistencies were also observed by other scientist when IPA was applied for cancer proteome analysis [ 58 – 63 ].…”

Characteristic of Ultrastructure of Mice B16 Melanoma Cells under the Influence of Different Lighting Regimes

“…A number of studies showed an increase in “spare respiratory capacity” of melanoma cells—the mitochondrial capacity to meet extra energy requirements, beyond the basal level, which occurred in response to acute cellular stress or heavy workload [ 45 ]. In particular, this occurs due to activation of PGC1α-dependent mitochondrial biogenesis, regulation of activity of enzymes involved in the respiratory chain by Sirtuin-3 directly and through an increased activity of the antioxidant enzyme SOD2, required for maintenance of spare respiratory capacity [ 46 , 47 ].…”