SGK1-regulated metabolism: key for the survival of cells detached from the extracellular matrix

ABSTRACT

Cells that lack attachment to the extracellular matrix (ECM) experience metabolic defects that can lead to caspase-independent cell death. Recently, we discovered that serum and glucocorticoid kinase-1 (SGK1) plays a critical role in the regulation of glucose metabolism, the promotion of energy production, and ultimately the survival of ECM-detached cells.

Abbreviations: ECM, extracellular matrix; SGK1, serum and glucocorticoid kinase-1; ROS, reactive oxygen species; CCCP, cyanide m-chlorophenyl hydrazine; PPP, pentose phosphate pathway; G3P, glyceraldhyde-3-phosphate; shRNA, short hairpin RNA; TCA, tricarboxylic acid

Loss of integrin-mediated attachment to the extracellular matrix (ECM) is well-known to initiate numerous cellular alterations that can lead to cell death.^1,2 Perhaps the most well studied death program in this context is anoikis, which is characterized by caspase-dependent cell death in response to disengagement of integrin receptors with ECM proteins.³ However, it has been observed that ECM-detached cells can also be killed through caspase-independent mechanisms. We have previously demonstrated that ECM-detachment causes fundamental changes in cell metabolism that compromise cell viability in an anoikis-independent fashion.^4–7 These metabolic changes include loss of glucose uptake, reduction in ATP production, and an elevation in the production of reactive oxygen species (ROS). The precise molecular mechanisms governing the relationship between metabolic reprogramming, ECM-detachment, and cell viability remain poorly understood.

Our previous studies revealed that the activation of serum and glucocorticoid kinase-1 (SGK1) could promote ATP generation and cell survival as downstream of HRAS activation in ECM-detached conditions.⁶ However, the mechanism by which SGK1 controlled ATP production was not delineated nor was the importance of SGK1 for the regulation of metabolism in the absence of activating HRAS mutations. Accumulating evidence has revealed that SGK1 is functionally important in the progression of multiple cancers.⁸ As such, in our recent study (see summary in Figure 1),⁹ we sought to ascertain the role that SGK1 plays in the regulation of metabolism in ECM-detached cancer cells with distinct oncogenic drivers. To begin, we introduced constitutively active SGK1 (S422D) into multiple cancer cell lines and found that SGK1 could robustly promote ATP generation in ECM-detached (but not ECM-attached) conditions. Similarly, using pharmacological inhibition or lentiviral transduction of short hairpin RNA (shRNA), we reduced the activity or expression of SGK1 in each cell line and found that SGK1 activity is necessary for ATP generation in ECM-detached (but not ECM-attached) conditions. Similarly, when we challenged cells to grow in an anchorage-independent fashion, we found that SGK1 is both sufficient and necessary for growth in soft agar. SGK1 has also been shown to function as an effector of the PIK3CA (hereafter referred to as PI(3)K) signaling pathway and shares overlap in function and sequence identity with AKT1 (hereafter referred to as AKT), the archetypical PI(3)K effector. However, when cells were engineered to express constitutively active AKT (through stable expression of myristoylated-AKT), an ECM-detachment-specific capacity of cells to produce ATP at high levels was not observed. Taken together, these data support an important regulatory role of SGK1 in ATP production during ECM-detachment that can facilitate anchorage-independent growth.

Figure 1.

SGK1-mediated ATP production in ECM-detached cells. In extracellular matrix (ECM)-detached conditions, serum and glucocorticoid kinase-1 (SGK1) activity leads to elevated transcription of the GLUT1 gene and subsequently to higher uptake of glucose (represented by green stars) into the cell. Thereafter, glucose-mediated carbon flux is directed through both glycolysis and the pentose phosphate pathway (PPP). From the non-oxidative arm of the PPP, glyceraldehyde-3-phosphate (G3P) is returned to the glycolysis where it is necessary for ATP generation and anchorage-independent growth

Abbreviations: G6PDH: glucose-6-phosphate dehydrogenase; TKT: transketolase.

To assess the molecular mechanisms by which SGK1 mediates ATP generation during ECM-detachment and anchorage-independent growth, we investigated the capacity of ECM-detached cells to take up glucose uptake in the presence or absence of SGK1 activation. Interestingly, we found that SGK1 activation could lead to a substantial elevation in glucose uptake in ECM-detached cells. Furthermore, we discovered that SGK1 (but not AKT) activation promotes an elevation in the transcription of GLUT1 and that GLUT1 inhibition abrogated the SGK1-mediated elevation in glucose uptake in ECM-detached cells. Given the capacity of SGK1 to promote the entry of glucose into the cells, we sought to determine the bioenergetic pathway(s) involved in facilitating SGK1-mediated ATP production and anchorage-independent growth. As expected, we discovered that glycolytic flux is important for the SGK1-mediated ATP generation during ECM-detachment. However, we were surprised to find that the addition of cell permeable pyruvate could not rescue ATP generation when SGK1 was inhibited. Given that pyruvate is typically metabolized in the tricarboxylic acid (TCA) cycle to produce ATP, we reasoned that the mitochondria were unimportant for SGK1-mediated ATP production. To further assess this possibility, we treated ECM-detached cells expressing constitutively active SGK1 with cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupler. Despite a reduction in ATP in control cells treated with CCCP, CCCP treatment of ECM-detached cells expressing constitutively active SGK1 did not alter ATP production. Furthermore, we used an additional, orthogonal approach to interrogate the role of the mitochondria in SGK1-mediated ATP production. We engineered HEK293T cells (expressing constitutively active SGK1) to inducibly maintain mitochondrial membrane potential while simultaneously lacking TCA cycle function.¹⁰ Following doxycycline-mediated ablation of TCA cycle activity in these cells, we observed that SGK1 (S422D) (but not myristoylated-AKT) could robustly promote glucose uptake and ATP generation during ECM-detachment. In aggregate, these results demonstrate that SGK1-mediated ATP production in ECM-detached cells is independent of mitochondrial oxidative phosphorylation and strongly suggests that SGK1 activation causes ECM-detached cells to rely on glycolytic flux for their bioenergetic needs.

Interestingly, after conducting stable isotope labeling to trace the fate of 1,2-¹³C glucose in cells expressing constitutively active SGK1, we found that intermediates of the pentose phosphate pathway (PPP) were also elevated to a substantial degree. Our previous studies had unveiled links between enhanced PPP flux, concomitant NADPH production, and diminished levels of ROS, ATP generation, and cell survival.⁷ Indeed, both pharmacological inhibition and shRNA of enzymes in the PPP (glucose-6-phosphate dehydrogenase (G6PDH) and transketolase (TKT)) led to a decrease in ATP generation during ECM-detachment and diminished anchorage-independent growth in cells expressing SGK1 (S422D). Surprisingly, the necessity of PPP flux for ATP generation in cells with activated SGK1 did not involve ROS mitigation. Instead, we determined that glyceraldhyde-3-phosphate (G3P) production by the PPP is critical for both ATP production and anchorage-independent growth in ECM-detached cells with SGK1 activation. In support of this, both exogenous addition of G3P and activation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rescued ATP generation and anchorage-independent growth in cells with PPP or SGK1 inhibition.

Here, we report that SGK1 activity is both necessary and sufficient for robust ATP generation during ECM-detachment and for anchorage-independent growth. Broadly speaking, our findings suggest that the status of integrin-mediated attachment to ECM can influence the capacity of signal transduction to alter cell metabolism and to impact cell death programs. Moreover, our findings suggest that ECM-detached cells are uniquely dependent on SGK1 signaling for the promotion of glucose-mediated carbon flux into multiple metabolic pathways. Likewise, given that GADPH activation or G3P addition rescues the loss of ATP generation caused by SGK1 or PPP inhibition, our data suggest that the PPP may function as a carbon reservoir after the substantial elevation in SGK1-driven glucose uptake. As ECM-detached cells are known to encounter metabolic defects, this capacity to allow robust carbon flux into both glycolysis and the PPP could provide GAPDH with glycolysis and PPP-derived G3P as substrates. Although the dynamics of glucose flux in cells expressing constitutively active SGK1 require future study, our data suggest that the dual sources of G3P provided to GAPDH in the presence of SGK1 activation can ultimately facilitate the survival of cells in anchorage-independent conditions.

Funding Statement

Z.T. Schafer is supported by funds from the National Cancer Institute of the National Institutes of Health [R01CA262439]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by funds from the American Cancer Society [RSG-14-145-01], a research grant from Phi Beta Psi Sorority, funds from the Coleman Foundation (Chicago, IL), the Malanga Family Excellence Fund for Cancer Research, and the University of Notre Dame.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.