The reprogramming of cellular metabolism during oncogenesis has attracted considerable recent attention, even being named one of the ‘emerging' hallmarks of cancer (Hanahan and Weinberg, 2011
). The metabolic reprogramming that helps satisfy the voracious appetite of tumor cells for biosynthetic precursors can also render cells exquisitely sensitive to nutrient deprivation (e.g., glucose and glutamine; Elstrom et al, 2004
; Yuneva et al, 2007
; Aykin-Burns et al, 2009
; Yang et al, 2009
). Here, we extend our understanding of this phenomenon with the phospho-tyrosine proteomic-based discovery that glucose deprivation provokes a systems-level positive feedback loop between ROS generation, PTPs, and TK signaling in cells dependent on glucose for survival (). Building on the unexpected observation that glucose withdrawal induces supra-physiological levels of phospho-tyrosine signaling, our systems-level feedback amplification loop model integrates the observations that (a) glucose withdrawal induces oxidative stress (Spitz et al, 2000
; Aykin-Burns et al, 2009
) and can activate diverse intracellular kinases including ERK, JNK, and Lyn (Lee et al, 1998b
; Blackburn et al, 1999
), (b) focal adhesions and RTKs serve as sites of NOX-mediated ROS generation (Lee et al, 1998a
; Wu et al, 2005
; Diaz et al, 2009
), and (c) ROS can inhibit PTPs, inducing further TK signaling (Lee et al, 1998a
; Mahadev et al, 2001
; Meng et al, 2002
). Taken together, this systems perspective reveals that glucose deprivation activates a positive feedback amplification loop, as indicated by supra-physiological levels of phospho-tyrosine signaling, until ROS accumulate above a toxicity threshold resulting in cell death.
Figure 8 Glucose withdrawal activates a positive feedback loop resulting in supra-physiological phospho-tyrosine signaling and ROS-mediated cell death. In cells dependent on glucose for survival, glucose and pyruvate deprivation induces oxidative stress driven (more ...)
Notably, the systems-wide positive feedback loop described here also functions in localized, subcellular contexts. A similar feedback amplification loop involving signaling, ROS, and PTPs occurs physically proximal to RTKs where EGF (Lee et al, 1998a
) and insulin (Mahadev et al, 2001
) induce oxidative inhibition of PTP-1B to promote signaling. Related positive feedback loops also occur at T-cell receptor signaling complexes (Kwon et al, 2010
) and focal complexes during endothelial cell migration (Wu et al, 2005
) and the formation of invadopodia (Diaz et al, 2009
). Here, we have demonstrated that a metabolic perturbation (i.e., glucose withdrawal) initiates a positive feedback amplification loop driven by NOX- and mitochondria-derived ROS generation resulting in cell-wide consequences on phospho-tyrosine signaling () and PTP activity (), ultimately resulting in cell death. In spatially localized contexts such as invadopodia or T-cell receptor signaling complexes, positive feedback is quickly dampened by reduction and re-activation of oxidized PTPs. However, in the case of metabolic deficiency, cells are unable to maintain redox homeostasis, perhaps due to depletion of cellular pools of NADPH (Ahmad et al, 2005
), which drives the amplification of ROS until a cellular toxicity threshold is breached and cells undergo ROS-mediated cell death. Thus, the ROS-PTP-TK positive feedback amplification loop set in motion by glucose withdrawal resembles positive feedback loops that are quickly dampened under normal, nutrient-rich conditions.
This work highlights the emerging concept of systems integration between oncogenic signaling networks and metabolism. For example, constitutively active oncogenic kinases, including myristoylated Akt and the activated EGFR mutant EGFRvIII, can promote aerobic glycolysis and lipogenesis, respectively (Elstrom et al, 2004
; Guo et al, 2009
). Conversely, glucose metabolism can influence signaling, such as the requirement of the hexosamine biosynthetic pathway for signaling through IL3-RA (Wellen et al, 2010
). Here, our model demonstrates the bidirectional interactions between signaling and metabolism. Our model predicts that either reduced TK signaling or increased PTP activity at sites of ROS generation (e.g., NOX complexes and mitochondria) should protect cells from glucose withdrawal-induced cell death. However, our data and model suggest that inhibition of a single kinase or overexpression of a single PTP is likely insufficient to rescue cells from glucose withdrawal-induced cell death precisely because cell death is controlled by a systems
-level positive feedback loop that simultaneously activates and inhibits multiple TKs and PTPs, respectively. Indeed, overexpression of PTEN, which functions as both a lipid and PTP (Myers et al, 1997
) and can undergo oxidative inactivation by RTK-induced NOX1 activity (Kwon et al, 2004
; Boivin et al, 2008
), only partially dampened the positive feedback loop in U87 cells (). The diminished activity of PTPs following glucose starvation () is also consistent with the observation by us and others that serine/threonine MAPK signaling is induced by glucose withdrawal (; Lee et al, 1998b
; Blackburn et al, 1999
), as dual-specificity phosphatases, which dephosphorylate MAPK enzymes, can be inhibited by oxidation of the catalytic cysteine residue (Kamata et al, 2005
). Our observations thus add to the growing evidence supporting the systems-level integration of metabolism and signaling homeostasis.
The integrated nature of the ROS-PTP-TK positive feedback loop described here offers an opportunity for therapeutic intervention. Indeed, combinatorial activation of the ROS-PTP-TK positive feedback loop with glucose deprivation and PTP inhibition exhibited synergistic killing of U87 cells (). Because many types of cancer cells exhibit increased levels of ROS and weakened redox buffering compared with normal cells (Szatrowski and Nathan, 1991
; Toyokuni et al, 1995
), ROS-promoting small molecule drugs can be selectively toxic to cancer cells (Trachootham et al, 2006
; Raj et al, 2011
; Shaw et al, 2011
), Similarly, therapeutics targeting the metabolic inflexibility of cancer are being pursued for selective toxicity to tumor cells (Yuneva, 2008
; Simons et al, 2009
). Our results highlight the possibility of unifying these two concepts through the judicious selection of therapeutic cocktails targeted against redox homeostasis and the metabolic inflexibility of cancer.
In support of this hypothesis, combinations of the glycolytic inhibitor 2-deoxyglucose and redox modulators (e.g., bulthione sulfoximine and antimycin A) demonstrate enhanced cytotoxicity compared with either agent alone (Andringa et al, 2006
; Fath et al, 2009
). Alternatively, it may be possible to alter the redox balance of tumor cells using physiological signals such as fasting, which can slow the growth of tumor xenografts (Kalaany and Sabatini, 2009
) and enhance the efficacy of high-dose chemotherapy in mouse models (Lee et al, 2012
). In light of our data demonstrating that glucose withdrawal-induced phospho-tyrosine signaling is driven by focal adhesions, it is interesting to note that normal epithelial cells exhibit loss of glucose transporters and oxidative stress following detachment from extracellular matrix (Schafer et al, 2009
). Thus, the integrated nature of metabolism, redox homeostasis, and signaling may permit ‘synthetic lethal' therapeutic approaches with selective toxicity toward tumor cells.
Finally, we speculate that this glucose withdrawal-initiated positive feedback loop may regulate the survival of metabolically altered tumor cells and/or select for metabolic phenotypes in nutrient-limited environments in vivo
. Recently, it was demonstrated that low glucose media can select for colorectal cancer cells with mutated KRAS and increased GLUT1 expression (Yun et al, 2009
). In a manner similar to the hypoxic selection of cells with increased resistance to apoptosis (Graeber et al, 1996
), intermittent glucose deprivation could select against cells dependent on glucose for survival. Additionally, because hypoxia selects for cells with increased glycolysis (Kim et al, 2007
), it may be possible that hypoxic and therefore glycolytic tumors are preferentially sensitive to metabolic inhibition.
In summary, cellular redox homeostasis is maintained by the balance between ROS generation by required metabolic functions and ROS elimination. Likewise, signaling homeostasis is controlled by balancing kinase and phosphatase activity. Here, we demonstrate that the cellular microenvironment (i.e., nutrient availability) can alter the cellular redox balance, provoking a signaling-based positive feedback loop that amplifies ROS levels above a toxicity threshold resulting in cell death. This positive feedback loop demonstrates the complex, systems-level integration of homeostatic control mechanisms for metabolism (e.g., redox balance) and TK signaling (e.g., PTPs). Furthermore, this systems integration offers a scaffold for synergistic combinations of therapeutics targeting signaling, metabolism, and redox homeostasis.