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The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-β-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation.

We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.

Recently, we reported that cancer cells that recover from a potentially lethal damage gain new phenotypic features comprised of mitochondrial structural remodeling associated with increased glycolytic dependency and drug resistance. Here, we demonstrate that a subset of cancer cells, upon recovery from a potentially lethal damage, undergo dedifferentiation and express genes, which are characteristic of undifferentiated stem cells. While these cells are competent in maintaining differentiated progeny of tumor, they also exhibit transdifferentiation potential. Dedifferentiation is characterized by accumulation of hydrogen sulfide (H2S), which triggers up-regulation of nicotinamide phosphoribosyltransferase (Nampt) accompanied by changes in the redox state. The molecular events triggered by Nampt include elevated production of NAD+ and up-regulation of H2S producing enzymes, cystathionine beta synthase (CBS) and cystathionase (CTH) with 3-mercaptopyruvate sulfurtransferase (MST) being detectable only in 3D spheroids. Suppression of Nampt, or inactivation of H2S producing enzymes, all reduce H2S production and reverse the ability of cells to dedifferentiate. Moreover, H2S induced stem cell markers in parental cancer cells in a manner similar to that observed in damage recovered cells. These data suggest of existence of a positive feedback loop between H2S and Nampt that controls dedifferentiation in cancer cells that recover from a potentially lethal damage.