Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jun 19.
Published in final edited form as: Mol Cell. 2014 May 22;54(6):960–974. doi: 10.1016/j.molcel.2014.04.024

Gain-of-Function Mutant p53 Promotes Cell Growth and Cancer Cell Metabolism via Inhibition of AMPK Activation

Ge Zhou 1,*, Jiping Wang 1, Mei Zhao 1, Tong-Xin Xie 1, Noriaki Tanaka 1, Daisuke Sano 1, Ameeta A Patel 1, Alexandra M Ward 1, Vlad Sandulache 1, Samar A Jasser 1, Heath D Skinner 1, Alison Lea Fitzgerald 1, Abdullah A Osman 1, Yongkun Wei 2, Xuefeng Xia 3, Zhou Songyang 4,5, Gordon B Mills 6, Mien-Chie Hung 2, Carlos Caulin 1, Jiyong Liang 6, Jeffrey N Myers 1,7,*
PMCID: PMC4067806  NIHMSID: NIHMS590697  PMID: 24857548

SUMMARY

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function.

INTRODUCTION

Mutation of the TP53 tumor suppressor gene is one of the most frequent genetic alterations in cancer, including head and neck squamous cell carcinoma (HNSCC) (Agrawal et al., 2011; Stransky et al., 2011). Although mutation of the TP53 gene can result in loss of wild-type p53 (wtp53) function or exert a dominant-negative effect over the remaining wild-type allele, some mutated forms of p53 (mutp53s) can lead to a gain of oncogenic properties that promote tumor growth and progression. However, the mechanisms involved in mutp53 gain of function (GOF) remain relatively poorly understood (Oren and Rotter, 2010).

Metabolic alterations, particularly the metabolic reprogramming to aerobic glycolysis (i.e., the Warburg effect) and the reprograming of mitochondrial metabolism, represent a hallmark of cancer that contributes to malignant transformation as well as the growth and maintenance of tumors (Hanahan and Weinberg, 2011; Vander Heiden et al., 2009; Ward and Thompson, 2012). In vivo dynamic mechanisms such as phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian homolog of target of rapamycin (mTOR) and adenosine monophosphate (AMP)-activated protein kinase (AMPK) sense the cellular energy status and regulate the balance between anabolism [an adenosine triphosphate (ATP)-consuming process that leads to macromolecular synthesis ] and catabolism (a process that degrades marcomolecules to release energy through increased ATP production) (Deberardinis and Thompson, 2012). AMPK is a highly conserved heterotrimeric serine/threonine protein kinase complex composed of a catalytic α subunit and regulatory β and γ subunits. As a major cellular energy sensor and a master regulator of metabolic homeostasis, AMPK is sensitive to the cellular AMP:ATP and adenosine diphosphate:ATP ratios and is activated by metabolic stresses that inhibit ATP production or stimulate ATP consumption (Hardie et al., 2012). Once activated, AMPK stimulates catabolism while inhibiting anabolism. AMPK achieves these effects by targeting many downstream metabolic enzymes [e.g., acetyl-CoA carboxylase (ACC) and mTOR] and by phosphorylating transcription factors [e.g., sterol regulatory element-binding protein 1 (SREBP1)] or cofactors that regulate gene expression (Hardie et al., 2012; Mihaylova and Shaw, 2011).

Studies have shown that wtp53 can regulate many metabolic pathways, such as carbohydrate and lipid metabolism, ROS regulation and autophagy (Berkers et al., 2013; Goldstein and Rotter, 2012). Importantly, stimulation of AMPK leads to the phosphorylation and activation of wtp53 (Jones et al., 2005; Okoshi et al., 2008). However, it remains unclear whether wtp53 is the direct target of AMPK (Fogarty and Hardie, 2010; Hardie, 2011). Recently, AMPK was shown to promote the stability of wtp53 indirectly through phosphorylation and inactivation of MDMX (He et al., 2014) and the p53 deacetylase, SIRT1 (Lee et al., 2012). The activation of wtp53 by AMPK signaling is believed to establish a metabolic checkpoint to suppress the growth of cells under conditions of metabolic stress (Jones et al., 2005). Therefore, AMPK is considered a tumor suppressor (Faubert et al., 2013; Luo et al., 2010). Moreover, once activated, wtp53 can, in turn, increase AMPK activity through transcriptional activation of the gene encoding the β subunit of AMPK (Feng et al., 2007) and sestrin (Budanov and Karin, 2008), providing a positive feedback effect to AMPK function. This positive feedback between AMPK and wtp53 is believed to play an important role in tumor suppression.

The vast majority of mutant p53s arise from missense mutations that can cause significant alterations in tertiary structure (Xu et al., 2011) which, in turn, can cause changes in p53 function through altered protein-protein interactomes and/or altered regulation of gene expression, thereby contributing to mutp53 GOF properties (Freed-Pastor and Prives, 2012; Muller and Vousden, 2013; Solomon et al., 2012). Recently, mutp53s were also shown to regulate metabolic pathways, such as steroid metabolism, via regulation of the transcription factor SREBP (Freed-Pastor et al., 2012), a downstream target of AMPK that directly phosphorylates and inhibits SREBP activity (Li et al., 2011). In the current study, we show that AMPK signaling is inhibited by GOF mutp53s. Moreover, we show that GOF mutp53s, but not wtp53, preferentially bind to the AMPKα subunit and directly inhibit AMPK activation, which increases anabolic growth and contributes to the GOF properties of mutp53s.

RESULTS

mutp53s Gain Oncogenic Function to Promote Invasive Growth of HNSCC Cells Both In Vitro and In Vivo

To study the functional impact of TP53 mutations, we first selected several human tumor-derived HNSCC cell lines with various TP53 status (Figures 1A and 1B) with which to test the function of mutp53s. When treated with the DNA-damaging agent 5-fluorouracil (5-FU), all the cell lines with mutp53s appeared to have lost wtp53 transcriptional function, as shown by the lack of induction of expression of the wtp53 targets, MDM2 and p21 (Figure 1B). One HNSCC cell line tested, UMSCC1, was found to lack detectable endogenous p53 protein due to the instability of splice site mutants (Figures 1A, 1B, and S1). This cell line was then stably transfected with various mutp53s (Figure 1C) to evaluate whether they could confer GOF activity. Overexpression of mutp53s P151S, R175H, G245C and R282W in UMSCC1 cells promoted invasive cell growth, as characterized by the formation of larger tumor spheroids with longer migration and invasion distances in an inverted three-dimensional (3D) Matrigel culture when compared with retroviral vector pBabe (Figure 1D), whereas expression of the C-terminal truncation mutp53, E336X, did not significantly promote UMSCC1 cell growth in the same assay (Figure 1D).

Figure 1. mutp53s Gain Oncogenic Function to Promote Invasive Growth of HNSCC Cells.

Figure 1

Open in a new tab

(A) Schematic diagram of wtp53 and mutp53s identified in the HNSCC cell lines shown in (B).

(B) Immunoblotting (IB) analysis of lysates from HNSCC cell lines with wtp53 or mutp53s in response to 5-FU treatment (1 mM, 24 hr). Breast cancer cell line MCF-7 and HNSCC cell line HN30 with wtp53 were used as the positive controls.

(C) IB analysis of UMSCC1 cell lines stably overexpressing various mutp53s after retroviral transfection. Various HNSCC cell lines with corresponding endogenous mutp53s and UMSCC1 cells expressing retroviral vector pBabe were used as controls.

(D) Inverted in vitro 3D Matrigel culture of various UMSCC1 stable cell lines. Images were acquired using confocal microscopy every 40 µm from the bottom to the top of the Matrigel culture.

(E) IB analysis of control and lentiviral shp53RNA-transfected Detroit 562 and Tu138 cells. Ctr, control shRNA.

(F) Inverted in vitro 3D Matrigel culture of HNSCC cell lines Detroit 562 and Tu138. Quantitative data are represented as mean ± standard deviation (SD).

In further support of mutp53 GOF, we used lentivirus-mediated p53 short hairpin RNA (shp53RNA) interference to knock down the expression of endogenous mutp53s (Figure 1E). Suppression of endogenous mutp53 R175H and P151S expression in Detroit 562 and Tu138 cells, respectively, resulted in smaller tumor spheroids with less migration and invasion compared with the control (Figure 1F). To further support the idea that mutp53 P151S is a GOF mutant, we previously demonstrated that overexpression of mutp53 P151S in UMSCC1 cells led to increased tumor growth and decreased animal survival in a mouse orthotopic xenograft oral tumor model. Conversely, down-regulation of mutp53 P151S in Tu138 cells suppressed tumor growth and increased animal survival in the same model (Sano et al., 2011).

Collectively, our in vitro and in vivo results clearly demonstrated that certain mutp53s, such as P151S, R175H, G245C and R282W, gain oncogenic function to promote invasive cell growth, whereas the C-terminal truncation mutp53, E336X, exhibits limited or no significant GOF activities in UMSCC1 cells.

Overexpression of GOF mutp53s Inhibits AMPK Activation

The formation of larger tumor spheroids in 3D Matrigel by overexpression of GOF mutp53s in UMSCC1 cells could have been due to an increase in cell proliferation, cell growth (cell size), or both, which is strongly linked to mTOR signaling (Zoncu et al., 2011). Consistent with this, down-regulation of mutp53 P151S in Tu138 cells resulted in a slightly smaller cell size when cells were cultured under low-glucose culture condition (Figure S2A). Because wtp53 is involved in inhibiting mTOR signaling by modulating both positive (PI3K/AKT) and negative [liver kinase B1 (LKB1)/AMPK] upstream regulatory pathways of mTOR signaling (Feng and Levine, 2010), we then examined AKT and AMPK signaling in our stable cell lines expressing mutp53s. Interestingly, while no difference in AKT phosphorylation was observed in UMSCC1 stable cell lines (Figures 2D–2F), we noted that the induction of AMPKα Thr172 phosphorylation, which is required for AMPK kinase activation (Hawley et al., 1996), in response to glucose deprivation, 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), and metabolic stress (serum-free, glucose-free, and hypoxia) was inhibited in UMSCC1 cells expressing mutp53 R175H when compared with the control cell line lacking mutp53 expression (Figures 2A–2C). However, under the same conditions, AMPK activation was only minimally affected in cells expressing the C-terminal truncated, non-GOF mutp53 E336X (Figures 2A–2C).

Figure 2. Overexpression of GOF mutp53s Suppresses Activation of AMPK Signaling in Response to Energy Stress.

Figure 2

Open in a new tab

(A–F) IB analyses of various UMSCC1 stable cell lines in response to stress: glucose starvation (A, D-F), 1 mM AICAR (B), and metabolic stress [glucose-free, serum-free, and hypoxia (1% O2)] (C). In (B), cells were serum-starved overnight before AICAR treatment in the absence of serum.

(G and H) IB analyses of K-Ras–transformed or pBabe control p53(−/−) and p53(R172H/H) MEFs in response to glucose starvation. See also Figure S2.

We then examined whether other GOF mutp53s have similar inhibitory effects on AMPK signaling in UMSCC1 cells. Under sub-confluent culture condition two mutp53s, P151S and R175H, but not G245C and R282W, efficiently inhibited phosphorylation of AMPKα as well as ACC, a downstream target of AMPK (Winder et al., 1997), in response to glucose deprivation (Figures 2D). However, when cells were cultured under high-density cell culture condition, all GOF mutp53s, including G245C and R282W which did not inhibit AMPK activation in sub-confluent culture, inhibited AMPK activation in response to glucose deprivation when compared with the control (Figures 2E and S2B). Importantly, this inhibition appeared to be specific to GOF mutp53s, since overexpression of neither non-GOF mutp53 E336X nor wtp53 inhibited AMPK activation under the same condition (Figures 2A–2C, and 2F). To understand why all GOF mutp53s inhibited AMPK activation (AMPKα Thr172 phosphorylation), we further used quantitative reverse transcription PCR (RT-qPCR) and Western blotting to examine the possible role of transcription control involved in mutp53-mediated AMPK inhibition. Our results, however, showed no inhibition of mutp53s to expression of the subunits of AMPK and its upstream LKB1 kinase complexes (i.e., AMPKα/β/γ and LKB1/Mo25α/β/Stradα/β) that are directly involved in AMPK activation in response to energy stress (Hardie et al., 2012) in UMSCC1 cells (Figures 2D–2F, and S2C).

To further test the role of GOF mutp53s in AMPK signaling, we used mouse embryonic fibroblasts (MEFs) with or without mutp53R172H/H (equivalent to human mutp53R175H/H), which was shown to have GOF activity in a genetically engineered mouse model (Lang et al., 2004; Olive et al., 2004). Compared with control p53(−/−) MEF cells, mutp53R172H/H MEFs exhibited lower levels of induction of AMPK and ACC phosphorylation in response to glucose starvation under confluent culture condition (Figures 2G and 2H). Taken together, our results showed that unlike wtp53, which activates AMPK signaling (Budanov and Karin, 2008; Feng et al., 2007), GOF mutp53s can actually inhibit AMPK activation and AMPK signaling.

Down-Regulation of GOF mutp53s Enhances AMPK Activation Induced by Energy Stress

As further evidence that GOF mutp53s inhibit AMPK activation, knockdown of endogenous mutp53 P151S in Tu138 cells (Figures 3A and 3B), and mutp53 R175H in pharyngeal cancer Detroit 562 cells (Figure 3C) and breast cancer SKBR3 cells (Figure 3D) resulted in increased induction of AMPK and ACC phosphorylation in response to glucose deprivation or metabolic stress. In contrast, we did not observe any impact of down-regulation of wtp53 on AMPK activation in response to glucose deprivation in HN30 cells (Figure S3). Additionally, consistent with the role of AMPK as a negative regulator of the mTOR/S6 ribosomal protein pathway (Shackelford and Shaw, 2009), down-regulation of mutp53 P151S in Tu138 cells resulted in decreased phosphorylation of the S6 ribosomal protein in response to glucose deprivation (Figure 3E), presumably through enhanced AMPK signaling. Moreover, immunohistochemical (IHC) evaluation of orthotopically grown oral cancer xenografts derived from Tu138-control shRNA (Ctr) and Tu138-shp53 cells (Sano et al., 2011) confirmed that Tu138-shp53 tumors exhibit increased levels of phosphorylated AMPK (pAMPK) and ACC (pACC) compared with Tu138-Ctr tumors (Figure 3F).

Figure 3. Downregulation of Endogenous GOF mutp53s Enhances Activation of AMPK Signaling in Response to Energy Stress in HNSCC Cells and Breast Cancer SKBR3 cells.

Figure 3

Open in a new tab

(A–E) IB analyses of various stable cell lines in response to energy stress.

(F) IHC staining of p53, pAMPK, and pACC in tongue tumors of orthotopic xenografts derived from Tu138-Ctr and Tu138-shp53 cells. Tumors were harvested 30 days after 5 × 105 cells were injected into the tongues of nude mice. Shown is one representative staining of tumors from three different mice. Right panel, quantitative data are represented as mean ± SD.

(G and H) IB analyses of various p53 shRNA cell lines rescued by expression of shRNA-resistant mutp53s in response to glucose starvation.

(I) Expression of shRNA-resistant mutp53 R175H rescued the invasive growth of p53 shRNA Detroit 562 cells in an inverted 3D Matrigel culture. Relative migrating distance for each cell line from one of three representative experiments is shown in right panel. Data are represented as mean ± SD. See also Figure S3.

Finally, introducing an shRNA-resistant version of mutp53 R175H or P151S into Detroit 562 shp53 or Tu138 shp53 cells by retroviral infection partially restored mutp53 expression in these cells, which not only led to decreased levels of energy stress-induced pAMPK (Figures 3G and 3H) but also partly rescued the invasive growth and prevented the phenotypic regression of Detroit 562 shp53 cells in 3D Matrigel culture (Figure 3I). These results, taken together, further supported our hypothesis that GOF mutp53s negatively regulate AMPK activation and signaling and contribute to the invasive growth phenotype of HNSCC cells both in vitro and in vivo.

Energy Stress Induces the Interaction of GOF mutp53s and AMPK In Vivo

In vivo, AMPK is shuttled between the nucleus and cytoplasm (Kodiha et al., 2007). However, cytoplasm is where AMPK is activated by its upstream kinase in response to energy stress (Tsou et al., 2011). To investigate the mechanisms involved in mutp53- mediated AMPK inhibition, we first examined the subcellular localization, especially the cytoplasmic localization, of mutp53s in UMSCC1 cells. Immunofluorescent staining revealed that GOF mutp53 P151S was localized in both the nucleus and cytoplasm of UMSCC1 cells (Figure 4A). In contrast, mutp53 E336X, likely because of its lack of a C-terminal p53 nuclear export signal (residues 340–351) (Stommel et al., 1999), localized exclusively in the nucleus of UMSCC1 cells (Figure 4B). Since this mutp53 E336X could not inhibit AMPK activation in vivo (Figures 2A–2C), the subcellular localization of mutp53s (i.e. the cytoplasmic localization) might be critical for mutp53-mediated AMPK inhibition. Consistent with this notion, GOF mutp53 G245C, which could only efficiently inhibit AMPK activation in high-density cell culture (compare Figures 2E and 2D), was predominantly localized to the nucleus in low-density cell culture (Figure 4C). However, its increased cytoplasmic localization was seen when cell density was increased (Figure 4C), suggesting that the stress from high-density cell culture facilitates the cytoplasmic localization of this mutp53.

Figure 4. Energy Stress Induces the Interaction of AMPK and mutp53s In Vivo.

Figure 4

Open in a new tab

(A–C) Immunofluorescent stainings of mutp53s in UMSSC1 stable cells under different culture conditions as indicated. Hypoxia: 1% O2; normoxia (reoxygenation): 21% O2.

(D) IP and IB analyses of 293FT cells cotransfected with Myc-p53 R175H and GFP-AMPKα2 or control pEGFP vector.

(E) IP and IB analyses of 293FT cells cotransfected with HA-AMPKα2 and Myc-tagged wtp53 or mutp53s. wtp53 has a proline residue at codon 72, whereas all the mutp53s have an arginine residue at codon 72; thus, wtp53 and mutp53s migrate differently.

(F) IP and IB analyses of the interaction of AMPKα and mutp53 in different mutp53-expressing UMSCC1 stable cell lines. Cells were starved of glucose for 2 hr before being harvested for IP.

(G) IP and IB analyses of the interaction of endogenous AMPKα and mutp53 P151S in Tu138 cells under subconfluent normal culture condition.

(H) IP and IB analyses of the interaction of endogenous AMPKα/β and mutp53 R175H in Detroit 562 cells.

(I) IP and IB analyses of the interaction of endogenous AMPKα and mutp53 in HNSCC cell lines. Cells were starved of glucose for 2 hr before being harvested for IP. (J and K) IP and IB analyses of the interaction of endogenous mutp53 and AMPKα in HNSCC cell lines in response to energy stress.

(L) IP and IB analyses of the interaction of endogenous mutp53 R175H and AMPKα/β/γ in Detroit 562 cells in response to energy stress. See also Figure S4.

Next, we investigated whether mutp53s could interact with AMPK in vivo. To this end, we first transfected 293FT human embryonic kidney cells with green fluorescent protein (GFP)-tagged AMPKα2 and Myc-tagged mutp53 R175H. Our results showed that mutp53 R175H was strongly associated with the GFP-AMPKα2 complex but not with control GFP (Figure 4D). Similarly, when Myc-tagged GOF mutp53s P151S, R175H, G245C and R282W were transfected, they were all able to complex with hemagglutinnin (HA)-tagged AMPKα2 expressed in 293FT cells (Figures 4E, lanes 5–8, and Figure S4). In contrast, neither transfected wtp53 nor the nucleus-localized mutp53 E336X was able to complex with hemagglutinnin (HA)-tagged AMPKα2 in the same assay (Figure 4E, compare lanes 4 and 9 to lanes 5–8), suggesting that compared to wtp53 and E336X, GOF mutp53s (i.e., P151S, R175H, G245C and R282W) gain the capability to interact with AMPKα2 in 293FT cells.

The interaction of GOF mutp53s P151S, R175H, G245C, and R282W with endogenous AMPKα was also observed in UMSCC1 cell lines stably expressing these mutp53s (Figure 4F) and in different HNSCC cell lines expressing various endogenous mutp53s (Figures 4G–4K), whereas no interaction between AMPKα and wtp53 or nucleus-localized mutp53 E336X was detected under the same conditions (Figures 4F and 4I–4K). Our results further showed that although endogenous GOF mutp53s P151S and R175H weakly interacted with the endogenous AMPKα in Tu138 and Detroit 562 cells under normal culture conditions, the enhanced interaction between mutp53s and AMPKα could actually be induced by glucose deprivation (Figures 4J and 4K), suggesting that energy stress induces AMPKα–mutp53s interaction in vivo. Finally, our results confirmed that in the immunoprecipitated mutp53-AMPKα complex from Detroit 562 cells, the β and γ subunits of AMPK were also detected (Figures 4H and 4L), suggesting that GOF mutp53s interact not only with endogenous α subunit of AMPK, but also with endogenous AMPK(α/β/γ) holoenzyme in vivo.

Taken together, these results clearly showed that not only does the interaction between GOF mutp53s and AMPK (α/β/γ) occur in vivo but also that it is induced by energy stress.

mutp53s, but Not wtp53, Preferentially Bind to AMPKα and Inhibit AMPK Activation

To investigate whether mutp53s can directly interact with AMPKα, we performed glutathione S-transferase (GST) pull-down assays using either AMPKα2-6xHis expressed in bacteria (Figure 5B) or in vitro transcribed and translated AMPKα (Figures 5C and 5D) and purified bacterially expressed wild-type and mutant GST-p53 proteins (Figures 5A–5D). Similar to our in vivo results, the GST pull-down assay demonstrated that mutp53s were indeed able to directly interact with both AMPKα1 and AMPKα2 in vitro (Figures 5B–5D) and that both the DNA-binding domain (DBD) and the C-terminus of p53 were required for p53–AMPKα interaction (Figures 5A and 5B). Interestingly, it appeared that all the GST-p53 fusion proteins tested, including GST fusion proteins of wtp53 and mutp53 E336X, that did not interact with AMPKα in vivo (Figures 4E, 4F, and 4I–4K), interacted with AMPKα directly in vitro in a GST pull-down assay (Figure 5A–5D). Since p53 is a very sensitive molecule and one amino acid change often converts wtp53 into a mutp53 with a different conformation, the fusion protein tag may significantly affect the p53 molecule. To minimize the impact of the N-terminal GST tag (27KD) on p53 conformation and physical properties, we further purified and used C-terminal 6xHis (1KD)-tagged p53 proteins in an in vitro co- immunoprecipitation (IP) assay. The results indicated that wtp53 had a much weaker binding affinity to AMPKα2 than did GOF mutp53s (Figure 5E). This observation was confirmed with an enzyme-linked immunosorbent assay (ELISA)-based protein-binding assay (Figure S5A).

Figure 5. mutp53s Directly Bind to the AMPKα Subunit and Inhibit AMPK Activation by Upstream Kinases.

Figure 5

Open in a new tab

(A) Schematic presentation of wtp53 and mutp53 proteins used in the GST pull-down assay. DBD, DNA-binding domain.

(B) GST pull-down assay of the interaction between purified AMPKα2-6xHis and GST-wtp53/mutp53 proteins.

(C and D) GST pull-down assay of the interaction between in vitro translated 35S-labelled AMPKα1 (C) or AMPKα2 (D) and the same amounts of purified GST-wtp53/mutp53 proteins. Shown is a representative result that was conducted simultaneously for both AMPKα1 and AMPKα2. Relative quantitative data (mean ± SD) from two independent experiments were calculated from gel densitometry using NIH imaging software and are shown at top. The activity of R175H was used as the referent (100%).

(E) In vitro co-IP/IB assay using purified wt/mutp53-6xHis and AMPKα2-6xHis proteins.

(F and G) LKB1 (F) and TAK1 (G) in vitro kinase assays using 50 ng of purified GST-AMPKα1 as the substrate in the presence of the exact amount of bovine serum albumin (BSA) (1–4 µg) or various purified GST-wtp53/mutp53 proteins as indicated on Coomassie blue gel for each reaction (30 µl of total assay volume). Kinase assays were conducted in the presence of Xa factor to cleave the N-terminal GST tag of p53 proteins.

(H) N-terminus of mutp53 R175H is required for blocking AMPKα2-LKB1 interaction. In vitro His pull-down assay used different purified AMPKα2, LKB1, and p53 proteins as indicated. Results and experimental procedure are summarized on left. Pull-down assay was conducted in the presence of Xa factor.

(I) Linked LKB1-mediated AMPKα-Thr172 phosphorylation/AMPK activity assay. The experimental procedure is shown at left and the results at right. Kinase assay was conducted in the presence of Xa factor. Relative AMPK activity is represented as mean ± SD. See also Figure S5.

To test whether the direct binding of AMPKα can inhibit its activation (α-Thr172 phosphorylation) by upstream kinases, we carried out in vitro kinase assays using the active AMPK upstream kinase, LKB1 or TGF-β–activated kinase-1 (TAK1), and AMPKα1 or α2 substrates in the presence or absence of purified GST-tagged cleaved wtp53 or different mutp53s. Under these experimental conditions, AMPKα–Thr172 was efficiently phosphorylated by LKB1 or TAK1, and AMPKα phosphorylation was strongly inhibited by mutp53s, whereas no inhibition was observed when similar amounts of wtp53 or the N-terminal deletion Δ161 mutant were used (Figures 5F, 5G, and S5B).

Given that both GST-wtp53 and -Δ161 bound to AMPKα (Figures 5A–5D) but the GST-tagged cleaved wtp53 or Δ161 did not inhibit AMPKα Thr172 phosphorylation (Figures 5F and 5G), the binding itself may be insufficient to block the activation of AMPK and the inhibition of α-Thr172 phosphorylation by upstream kinases may require an N-terminally intact p53 with the “mutant molecular conformation”. In support of this notion, our in vitro pull-down assay using different purified proteins showed that although wtp53 and the mutants Δ161, R175H, and Δ161-R175H all bound to AMPKα2, only the GOF mutant R175H efficiently inhibited AMPKα2-LKB1 interaction (Figure 5H). Therefore, whereas DBD is required for p53-AMPKα binding (Figures 5A and 5B), the N-terminus of mutp53s is responsible for blocking AMPKα-LKB1 interaction (Figure 5H).

Finally, since active AMPK is a heterotrimeric serine/threonine protein kinase complex that contains not only an α subunit (catalytic subunit), but also regulatory β and γ subunits (Hardie et al., 2012), after demonstrating that mutp53s could bind to the α subunit and inhibit its Thr172 phosphorylation by upstream kinases (Figures 5A–5H), we investigated whether mutp53s can directly affect AMPK activation as well as its subsequent kinase activity. To test this idea, we first co-transfected 293FT with GFP-AMPK β1 and HA-AMPKα2, and then used Chromotek-GFP-Trap agarose beads to pull down AMPK (α/β/γ), which was further dephosphorylated by phosphatase PP2C. Subsequently, this dephosphorylated AMPK (inactive kinase) was used in a linked LKB-mediated AMPKα-Thr172 phosphorylation/AMPK activity assay in the presence or absence of different wtp53 and mutp53s (Figure 5I, left). The mutp53s R175H, G245C, P151S and E336X were also able to inhibit α-Thr172 phosphorylation and suppress activation of AMPK by LKB1, subsequently suppressing its kinase activity (Figure 5I, right). In contrast, wtp53 that bound to AMPKα weakly (Figure 5E) and the ΔDBD mutp53 that did not bind to AMPKα (Figure 5B) exhibited either marginal or no impact on α-Thr172 phosphorylation and AMPK activation by LKB1 (Figure 5I, right).

Taken together, our results demonstrated that mutp53 directly binds to the AMPKα subunit via the DBD while its N-terminus blocks the interaction between AMPKα and upstream kinases, leading to inhibition of AMPK activation and AMPK kinase activity.

Inhibition of AMPK Rescues Both Decreased Invasive Cell Growth and Impaired Aerobic Glycolytic Potential Associated with Loss of GOF mutp53s

Consistent with the tumor-suppressing role of AMPK, knockdown of AMPKα1 in p53-deficient UMSCC1 and PCI-13 cells promoted cell growth in 3D Matrigel (Figures S6A–S6D). To further test the role of inhibition of AMPK activation in the GOF mutp53-associated phenotype, we knocked down AMPKα in mutp53 down-regulated cells. While down-regulation of mutp53 P151S in Tu138 cells resulted in decreased S6 phosphorylation in response to glucose starvation, further knockdown of AMPKα2 in those cells by two independent shRNAs rescued the decreased phosphorylation of ribosomal protein S6 due to the loss of mutp53 P151S (Figure 6A).

Figure 6. Inhibition of AMPK Rescues the Decreased Invasive Cell Growth and Impaired Aerobic Glycolytic Potential Associated with Loss of GOF mutp53s.

Figure 6

Open in a new tab

(A) IB analysis of pAMPK, pACC, and pS6 ribosomal protein in various Tu138 stable cell lines in response to glucose deprivation. NT, non-target.

(B) Flow cytometry analysis of cell size of various Tu138 stable cell lines. Cells were cultured in 0.5 mM glucose Dulbecco’s modified Eagle’s medium (DMEM) overnight before being subjected to analysis.

(C) Impaired cell growth due to loss of GOF mutp53 in 3D Matrigel was rescued by inhibition of AMPK. Shown are representative images of 3D culture of Tu138 stable cell lines.

(D) IB analysis of various Tu138 stable cell lines in response to glucose deprivation.

(E) Inverted in vitro 3D Matrigel culture of various stable cell lines.

(F) Oil Red O staining of confluent p53(−/−) and p53(R172H/H) MEFs cultured in the presence of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine. Top: Oil Red O–stained dishes. Bottom: Staining was quantified by absorbance at OD495 nm. Data are means ± SD (n = 3).

(G and H) Glycolytic rate (ECAR) of various Detroit 562 (G) and Tu138 (H) stable cell lines in response to glucose stimulation. Cells were starved of glucose for 2 hr prior to initiation of experiment. Drugs were added at time points A (glucose, 25 mM), B (oligomycin, 1 µM), C (FCCP, 300 nM), and D (rotenone and antimycin, 100 nM). Experiments were conducted in the absence of sodium pyruvate. Data are presented as averages of 3–5 measurements. Error bars represent standard error of the mean (SEM). See also Figures S6 and S7.

Because ribosomal protein S6 is a critical downstream mediator of mTOR regulation of protein synthesis and cell growth (Zoncu et al., 2011), we expected that GOF mutp53s would enhance cell growth (i.e., cell size) through S6 activation. Flow cytometry analysis demonstrated that down-regulation of mutp53 P151S in Tu138 cells resulted in a slight but consistent decrease in the cell size of both G1 and G2/M cells cultured in low-glucose medium (0.5 mM glucose) (Figure 6B). More important, the decrease in cell size induced by the loss of mutp53 was rescued by knockdown of AMPKα2 expression in Tu138 shp53RNA cells (Figure 6B). Similarly, down-regulation of AMPKα2 in Tu138 shp53RNA and Detroit 562 shp53RNA cells rescued the impaired invasive cell growth associated with loss of mutp53s in an in vitro 3D Matrigel culture assay, which resulted in much larger and more disorganized tumor spheroids with longer migration/invasion distances (Figures 6C–6E, and S6E-S6H). Furthermore, introducing an shRNA-resistant version of AMPKα2 into mutp53- and AMPKα1&2–double knockdown Tu138 and Detroit 562 cells by retroviral infection (Figures 6D and S6G) reversed the phenotype, leading to smaller cell size (Figure S6I) and decreased invasive cell growth with less cell migration and invasion in an inverted 3D Matrigel culture (Figures 6E and S6H). Together, these results strongly suggested that AMPK is an important downstream target of GOF mutp53s and a critical mediator of the observed cell growth and invasive phenotype associated with mutp53 GOF.

Phosphorylation of ACC and SREBP-1c by AMPK leads to inhibition of ACC and SREBP-1c activity, resulting in increased fatty acid oxidation and decreased lipogenesis (Hardie and Pan, 2002; Li et al., 2011). Given the inhibitory role of GOF mutp53s on AMPK, we expected that GOF mutp53s would also regulate lipid metabolism. Consistent with this idea, p53(R172H/H) MEFs treated with a combination of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine, which stimulates lipogenesis (Esfandiari et al., 2007), exhibited much higher lipid levels than p53(−/−) MEFs did as evaluated by Oil Red O staining (Figure 6F).

Finally, we examined the role of mutp53s in aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), as measured by the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) (Marshall et al., 2011). Although down-regulation of mutp53 had no obvious impact on OXPHOS response (Figures S7A and S7B), it significantly impaired aerobic glycolytic capacity in response to glucose stimulation in both Detroit 562 and Tu138 cells (Figures 6G, 6H, S7C, and S7D). Furthermore, an shRNA-resistant mutp53 P151S rescued the impaired aerobic glycolytic capacity in Tu138 shp53RNA cells (Figure S7D). More important, the impaired aerobic glycolytic capacity due to the loss of mutp53 was partially rescued by further knockdown of AMPKα (Figures 6G, 6H, and S7E). However, this rescue was reversed by expression of an shRNA-resistant AMPKα2 in mutp53- and AMPKα1&2-double knockdown Tu138 cells, leading to decreased aerobic glycolytic capacity in response to glucose stimulation (Figure 6H).

Taken together, these results suggested that GOF mutp53s are involved in promoting the invasive growth and metabolism of cancer cells, such as lipid synthesis or accumulation and aerobic glycolysis, at least partly through the inhibition of AMPK.

GOF mutp53s Promote Tumor Growth and Inhibit AMPK Activation in Orthotopic Xenografts and GOF mutp53 Genetically Engineered Mouse Models of HNSCC

In support of the tumor-suppressing role of AMPK, down-regulation of AMPKα in p53-deficient PCI-13 cells (Figure S6A) resulted in increased tumor growth in an orthotopic mouse oral cancer model (Figure S7F). To further gain insight into mutp53 GOF activity in the same animal tumor model, we injected UMSCC1 stable cell lines expressing different wt and different mutp53s into the tongues of nude mice. GOF mutp53s R175H, G245C and R282W, but not mutp53 E336X, significantly accelerated tumor onset and promoted tumor growth when compared with wtp53 and pBabe control (Figures 7A and S7G). Moreover, while pBabe and wtp53 tumors appeared to grow in defined areas and exhibited a more differentiated morphology, GOF mutp53 tumors displayed a more aggressive phenotype and often infiltrated and invaded into adjacent tissues (Figure 7B). Consistently, IHC staining indicated that AMPK activation (AMPKα–Thr172 phosphorylation) was inhibited in tumors derived from UMSCC1 stable cell lines expressing GOF mutp53s R175H, G245C, and R282W when compared with the controls (Figure 7C).

Figure 7. GOF mutp53s Promote Tumor Growth and Inhibit AMPK activation in Orthotopic Xenografts and GOF mutp53 Genetically Engineered Mouse Model of HNSCC.

Figure 7

Open in a new tab

(A) Oral tumor growth of UMSCC1 stable cell lines expressing different wtp53 and mutp53s. 1× 105 cells/mouse were injected into the tongue of nude mouse. Error bar: SD.

(B) Hematoxylin and eosin staining of tumor samples from UMSCC1 wtp53 and mutp53 stable cell lines injected into the tongues of nude mice.

(C) IHC staining of p53 and pAMPK in tongue tumor samples from an orthotopic mouse model of UMSCC1 wtp53 and mutp53 stable cell lines. Error bar: SD. *p<0.0001, n=4.

(D) IHC staining of p53 and pAMPK in papilloma samples from mice with the indicated genotypes. Error bar: SD. *p<0.0001, n=4.

(E) A working model for GOF mutp53s, in which wtp53 functions indirectly downstream of AMPK and GOF mutp53s function directly upstream of AMPK and inhibit its activation, leading to an impaired metabolic checkpoint and increased anabolic tumor growth and progression. See also Figure S7.

Finally, using a genetically defined mouse model in which oral papillomas arise from the activation of endogenous oncogenic K-ras, in the presence or absence of additional GOF mutp53R172H or loss-of-function (p53−/−) mutations (Acin et al., 2011), we evaluated whether GOF mutp53s modulate AMPK activation. pAMPK was highly expressed in the basal and suprabasal layers of the papilloma of p53(−/−) tumors (black arrows), whereas in GOF mutp53R172H/− papilloma, mutp53 happened to be highly expressed in the same layers, but with decreased levels of pAMPK (red arrows) (Figure 7D), suggesting that expression of mutp53 R172H in tumors inhibits AMPK activation, and thereby promotes tumor progression.

DISCUSSION

In the current study, we have shown that GOF mutp53s negatively regulate the metabolic effects of AMPK signaling, leading to increased lipid production, aerobic glycolysis, and invasive cell growth. Moreover, our results demonstrated that GOF mutp53s, through their DBDs, preferentially bind to the AMPKα subunit whereas their N-termini block AMPKα-LKB1 interaction, leading to inhibition of AMPK activation. On the basis of these results, we propose a working model whereby mutp53s can gain functional activity (Figure 7E): GOF mutp53s with high AMPKα-binding affinity gain oncogenic function to promote cell growth and cancer cell metabolism through direct inhibition of AMPK activation.

The majority of p53 missense mutations occur in the DBD, which usually leads to loss of the specific DNA-binding activity of p53 as a transcription factor that directly regulates gene expression. In spite of this, while missense mutations disrupt the specific p53 DNA-binding activity, they often cause significant alterations in p53 tertiary structure (Xu et al., 2011) which, in turn, can cause changes in p53 function through altered protein-protein interactomes and/or altered regulation of gene expression, contributing to mutp53 GOF properties (Freed-Pastor and Prives, 2012; Muller and Vousden, 2013; Solomon et al., 2012). As an excellent example, our results demonstrated a difference between wtp53 and mutp53s in terms of their ability to bind AMPKα and inhibit AMPK activation both in vitro and in vivo (Figures 2F, 4E, 4F, 4J, 5E–5I, 7C, and S3–S5), suggesting an important molecular mechanism for the difference between mutp53s and wtp53 in their abilities to interact with other proteins, regulate gene expression, and exert GOF properties.

Most mutp53 GOF mechanisms that have been proposed are involved in transcriptional control of gene expression, in which mutp53s function as either transcriptional coactivators or transcriptional corepressors to control the transcriptional machinery that regulates gene expression (Freed-Pastor and Prives, 2012; Muller and Vousden, 2013; Solomon et al., 2012). Among many possible GOF mechanisms, dominant-negative effects over other p53 family members, p63 and p73, were proposed (Li and Prives, 2007). Interestingly, TAp63 was reported to transcriptionally activate LKB1 and AMPKα2 expression in mouse fat, liver and muscle in a TAp63-knockout mouse model (Su et al., 2012), raising the possibility that GOF mutp53s inhibit LKB1 and AMPKα gene expression through dominant-negative effects over TAp63. However, while our evidence demonstrated that GOF mutp53s inhibit AMPK activation (Figures 2–7), our results using both real-time reverse-transcriptase PCR (Figure S2C) and Western blotting (Figures 2D–2F and 3C) revealed that expression of mutp53s had no significant impact on the expression of either LKB1 or AMPKα or on other subunits of LKB1 and AMPK kinases in HNSCC cells. Instead, all our evidence has indicated that GOF mutp53s have a direct role in AMPK activation (Figures 4 and 5).

As another piece of evidence supporting the transcription-independent role of GOF mutp53s, our results showed that although the C-terminal-truncated mutp53 E336X exhibited both AMPKα-binding and -inhibitory activity in vitro (Figures 5A–5D, 5F, 5G, and 5I), it failed to efficiently bind AMPKα (Figures 4E, 4F, 4I, and 4K) or inhibit AMPK activation (Figures 2A–2C) in vivo and also failed to promote tumor cell growth (Figures 1D and S7G). The fact that E336X localized exclusively in the nucleus (Figure 4B) suggested that the cytoplasmic localization of mutp53s is also important for their GOF activities. Consistent with this notion, a previous study reported that some mutp53 proteins localize in the cytoplasm and inhibit autophagy (Morselli et al., 2008). More important, our results showed that like AMPK, whose subcellular localization (e.g. the cytoplasmic localization) is influenced by cell density (Kodiha et al., 2007), the subcellular localization of mutp53s was subjected to the regulation of cell culture conditions such as cell density (Figure 4C). This finding may be important to understanding the biological functions of mutp53s.

AMPK was recently identified to negatively regulate the Warburg effect through inhibition of the hypoxia-induced factor 1 (HIF1) pathway (Faubert et al., 2013). Therefore, the inhibition of AMPK by GOF mutp53s, which relieves the suppression of HIF1 by AMPK, is expected to increase HIF1 protein expression and thus lead to increased glucose influx and glycolysis. Indeed, mutant p53s have been shown to promote HIF1 expression (Kamat et al., 2007) and HIF1 has been found to be required for thymic lymphomas to develop in a p53-mutant mouse model (Bertout et al., 2009).

In summary, our evidence strongly supports a transcription-independent mechanism that is important to the GOF activity of mutp53s. Moreover, since AMPK is a master cellular energy sensor and a regulator of metabolic homeostasis (Hardie, 2007), our demonstration that mutp53s are actively involved in the regulation of cancer metabolism through direct inhibition of AMPK further extends our understanding of the molecular mechanisms of mutp53s in cancers. Our findings also provide an excellent example of how a gene mutation in tumor cells can transform an agonistic relationship (i.e., wtp53 activating AMPK signaling) into an antagonistic relationship (i.e., mutp53 inhibiting AMPK). In this case, p53 mutation transforms a signaling network with tumor-suppressing functions into a network with oncogenic potential to promote tumor growth and progression.

EXPERIMENTAL PROCEDURES

See Supplemental Experimental Procedures.

Establishment of Stable Cell Lines Over- or Underexpressing Target Genes

Stable cell lines overexpressing a mutp53 or K-ras were obtained from pooled cells after puromycin (1–2 µg/ml) selection of retrovirus-infected cells. Lentiviral shRNA expression systems were used to stably knockdown mutp53 or AMPKα expression.

In Vitro Inverted Matrigel Culture

A total of 40,000 cells, mixed and embedded in 50 µl of BD-Matrigel (#354230; BD Biosciences), were layered on the surface of a BD BioCoat control insert (#354578; BD Biosciences) and incubated at 37°C for 30–45 min, allowing the Matrigel to solidify. An additional 200 µl of Matrigel was layered on the surface of the solidified Matrigel. When all the Matrigel was solidified, inserts were placed in a 24-well culture plate with 0.1 ml of serum-free culture medium in each lower chamber. On the upper side of the inserts, 0.3–0.5 ml of culture medium containing 20% fetal bovine serum (FBS) and 50 ng/ml epidermal growth factor (EGF) was added (see also Supplemental Experimental Procedures).

Glycolysis and OXPHOS Measurements

Glycolysis and OXPHOS were measured as ECAR and OCR, respectively, using Seahorse XF24 extracellular flux analyzers according to the manufacturer’s instructions.

Supplementary Material

01
02

Highlights.

  • Mutant p53 gains oncogenic function

  • Gain-of-function mutant p53 inhibits AMPK activation

  • Gain-of function mutant p53 regulates cancer cell metabolism via AMPK

  • Mutant p53 gains oncogenic function in a transcription-independent manner

ACKNOWLEDGMENTS

We thank Drs. Guillermina Lozano and Yoko Takahashi for providing p53(−/−) and p53(R172H/H) MEFs and SKBR3, UMSCC1, HN30, and Detroit 562 stable cell lines. We also thank Drs. Kun-liang Guan, Jianhua Yang, and Xiaolu Yang for providing the plasmids. This research was supported by the University Cancer Foundation via the Institutional Research Grant program at The University of Texas MD Anderson Cancer Center to G. Zhou and by NIH grants RO1 DE015344 to C. Caulin, CA133249 and GM095599 to Z. Songyang, and RO1 DE14613, P50CA097007, and CA016672 to J.N. Myers. The authors declare no competing financial interests.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  1. Acin S, Li Z, Mejia O, Roop DR, El-Naggar AK, Caulin C. Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras. J Pathol. 2011;225:479–489. doi: 10.1002/path.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, Fakhry C, Xie TX, Zhang J, Wang J, et al. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 2011;333:1154–1157. doi: 10.1126/science.1206923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkers CR, Maddocks OD, Cheung EC, Mor I, Vousden KH. Metabolic regulation by p53 family members. Cell Metab. 2013;18:617–633. doi: 10.1016/j.cmet.2013.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertout JA, Patel SA, Fryer BH, Durham AC, Covello KL, Olive KP, Goldschmidt MH, Simon MC. Heterozygosity for hypoxia inducible factor 1alpha decreases the incidence of thymic lymphomas in a p53 mutant mouse model. Cancer Res. 2009;69:3213–3220. doi: 10.1158/0008-5472.CAN-08-4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Budanov AV, Karin M. p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell. 2008;134:451–460. doi: 10.1016/j.cell.2008.06.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deberardinis RJ, Thompson CB. Cellular metabolism and disease: what do metabolic outliers teach us? Cell. 2012;148:1132–1144. doi: 10.1016/j.cell.2012.02.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Esfandiari F, You M, Villanueva JA, Wong DH, French SW, Halsted CH. S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet. Alcohol Clin Exp Res. 2007;31:1231–1239. doi: 10.1111/j.1530-0277.2007.00407.x. [DOI] [PubMed] [Google Scholar]
  8. Faubert B, Boily G, Izreig S, Griss T, Samborska B, Dong Z, Dupuy F, Chambers C, Fuerth BJ, Viollet B, et al. AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo. Cell Metab. 2013;17:113–124. doi: 10.1016/j.cmet.2012.12.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feng Z, Hu W, de Stanchina E, Teresky AK, Jin S, Lowe S, Levine AJ. The regulation of AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR pathways. Cancer Res. 2007;67:3043–3053. doi: 10.1158/0008-5472.CAN-06-4149. [DOI] [PubMed] [Google Scholar]
  10. Feng Z, Levine AJ. The regulation of energy metabolism and the IGF-1/mTOR pathways by the p53 protein. Trends Cell Biol. 2010;20:427–434. doi: 10.1016/j.tcb.2010.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fogarty S, Hardie DG. Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer. Biochim Biophys Acta. 2010;1804:581–591. doi: 10.1016/j.bbapap.2009.09.012. [DOI] [PubMed] [Google Scholar]
  12. Freed-Pastor WA, Mizuno H, Zhao X, Langerod A, Moon SH, Rodriguez-Barrueco R, Barsotti A, Chicas A, Li W, Polotskaia A, et al. Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway. Cell. 2012;148:244–258. doi: 10.1016/j.cell.2011.12.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Freed-Pastor WA, Prives C. Mutant p53: one name, many proteins. Genes Dev. 2012;26:1268–1286. doi: 10.1101/gad.190678.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldstein I, Rotter V. Regulation of lipid metabolism by p53 - fighting two villains with one sword. Trends Endocrinol Metab. 2012;23:567–575. doi: 10.1016/j.tem.2012.06.007. [DOI] [PubMed] [Google Scholar]
  15. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]
  16. Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev. 2011;25:1895–1908. doi: 10.1101/gad.17420111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hardie DG, Pan DA. Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase. Biochem Soc Trans. 2002;30:1064–1070. doi: 10.1042/bst0301064. [DOI] [PubMed] [Google Scholar]
  18. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012;13:251–262. doi: 10.1038/nrm3311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem. 1996;271:27879–27887. doi: 10.1074/jbc.271.44.27879. [DOI] [PubMed] [Google Scholar]
  20. He G, Zhang YW, Lee JH, Zeng SX, Wang YV, Luo Z, Dong XC, Viollet B, Wahl GM, Lu H. AMP-activated protein kinase induces p53 by phosphorylating MDMX and inhibiting its activity. Mol Cell Biol. 2014;34:148–157. doi: 10.1128/MCB.00670-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell. 2005;18:283–293. doi: 10.1016/j.molcel.2005.03.027. [DOI] [PubMed] [Google Scholar]
  22. Kamat CD, Green DE, Warnke L, Thorpe JE, Ceriello A, Ihnat MA. Mutant p53 facilitates pro-angiogenic, hyperproliferative phenotype in response to chronic relative hypoxia. Cancer Lett. 2007;249:209–219. doi: 10.1016/j.canlet.2006.08.017. [DOI] [PubMed] [Google Scholar]
  23. Kodiha M, Rassi JG, Brown CM, Stochaj U. Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK-->ERK1/2 pathway. Am J Physiol Cell Physiol. 2007;293:C1427–C1436. doi: 10.1152/ajpcell.00176.2007. [DOI] [PubMed] [Google Scholar]
  24. Lang GA, Iwakuma T, Suh YA, Liu G, Rao VA, Parant JM, Valentin-Vega YA, Terzian T, Caldwell LC, Strong LC, et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell. 2004;119:861–872. doi: 10.1016/j.cell.2004.11.006. [DOI] [PubMed] [Google Scholar]
  25. Lee CW, Wong LL, Tse EY, Liu HF, Leong VY, Lee JM, Hardie DG, Ng IO, Ching YP. AMPK promotes p53 acetylation via phosphorylation and inactivation of SIRT1 in liver cancer cells. Cancer Res. 2012;72:4394–4404. doi: 10.1158/0008-5472.CAN-12-0429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li Y, Prives C. Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function? Oncogene. 2007;26:2220–2225. doi: 10.1038/sj.onc.1210311. [DOI] [PubMed] [Google Scholar]
  27. Li Y, Xu S, Mihaylova MM, Zheng B, Hou X, Jiang B, Park O, Luo Z, Lefai E, Shyy JY, et al. AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metab. 2011;13:376–388. doi: 10.1016/j.cmet.2011.03.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Luo Z, Zang M, Guo W. AMPK as a metabolic tumor suppressor: control of metabolism and cell growth. Future Oncol. 2010;6:457–470. doi: 10.2217/fon.09.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marshall M, van der Windt D, Nicholls E, Myers H, Dziedzic K. Radiographic thumb osteoarthritis: frequency, patterns and associations with pain and clinical assessment findings in a community-dwelling population. Rheumatology (Oxford) 2011;50:735–739. doi: 10.1093/rheumatology/keq371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011;13:1016–1023. doi: 10.1038/ncb2329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morselli E, Tasdemir E, Maiuri MC, Galluzzi L, Kepp O, Criollo A, Vicencio JM, Soussi T, Kroemer G. Mutant p53 protein localized in the cytoplasm inhibits autophagy. Cell Cycle. 2008;7:3056–3061. doi: 10.4161/cc.7.19.6751. [DOI] [PubMed] [Google Scholar]
  32. Muller PA, Vousden KH. p53 mutations in cancer. Nat Cell Biol. 2013;15:2–8. doi: 10.1038/ncb2641. [DOI] [PubMed] [Google Scholar]
  33. Okoshi R, Ozaki T, Yamamoto H, Ando K, Koida N, Ono S, Koda T, Kamijo T, Nakagawara A, Kizaki H. Activation of AMP-activated protein kinase induces p53-dependent apoptotic cell death in response to energetic stress. J Biol Chem. 2008;283:3979–3987. doi: 10.1074/jbc.M705232200. [DOI] [PubMed] [Google Scholar]
  34. Olive KP, Tuveson DA, Ruhe ZC, Yin B, Willis NA, Bronson RT, Crowley D, Jacks T. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell. 2004;119:847–860. doi: 10.1016/j.cell.2004.11.004. [DOI] [PubMed] [Google Scholar]
  35. Oren M, Rotter V. Mutant p53 gain-of-function in cancer. Cold Spring Harb Perspect Biol. 2010;2:a001107. doi: 10.1101/cshperspect.a001107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sano D, Xie TX, Ow TJ, Zhao M, Pickering CR, Zhou G, Sandulache VC, Wheeler DA, Gibbs RA, Caulin C, et al. Disruptive TP53 mutation is associated with aggressive disease characteristics in an orthotopic murine model of oral tongue cancer. Clin Cancer Res. 2011;17:6658–6670. doi: 10.1158/1078-0432.CCR-11-0046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer. 2009;9:563–575. doi: 10.1038/nrc2676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Solomon H, Buganim Y, Kogan-Sakin I, Pomeraniec L, Assia Y, Madar S, Goldstein I, Brosh R, Kalo E, Beatus T, et al. Various p53 mutant proteins differently regulate the Ras circuit to induce a cancer-related gene signature. J Cell Sci. 2012;125:3144–3152. doi: 10.1242/jcs.099663. [DOI] [PubMed] [Google Scholar]
  39. Stommel JM, Marchenko ND, Jimenez GS, Moll UM, Hope TJ, Wahl GM. A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. The EMBO journal. 1999;18:1660–1672. doi: 10.1093/emboj/18.6.1660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333:1157–1160. doi: 10.1126/science.1208130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Su X, Gi YJ, Chakravarti D, Chan IL, Zhang A, Xia X, Tsai KY, Flores ER. TAp63 is a master transcriptional regulator of lipid and glucose metabolism. Cell metabolism. 2012;16:511–525. doi: 10.1016/j.cmet.2012.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tsou P, Zheng B, Hsu CH, Sasaki AT, Cantley LC. A fluorescent reporter of AMPK activity and cellular energy stress. Cell Metab. 2011;13:476–486. doi: 10.1016/j.cmet.2011.03.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–1033. doi: 10.1126/science.1160809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell. 2012;21:297–308. doi: 10.1016/j.ccr.2012.02.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Winder WW, Wilson HA, Hardie DG, Rasmussen BB, Hutber CA, Call GB, Clayton RD, Conley LM, Yoon S, Zhou B. Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. J Appl Physiol. 1997;82:219–225. doi: 10.1152/jappl.1997.82.1.219. [DOI] [PubMed] [Google Scholar]
  46. Xu J, Reumers J, Couceiro JR, De Smet F, Gallardo R, Rudyak S, Cornelis A, Rozenski J, Zwolinska A, Marine JC, et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat Chem Biol. 2011;7:285–295. doi: 10.1038/nchembio.546. [DOI] [PubMed] [Google Scholar]
  47. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol. 2011;12:21–35. doi: 10.1038/nrm3025. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS590697-supplement-01.pdf (4.4MB, pdf)
02
NIHMS590697-supplement-02.pdf (372.7KB, pdf)

RESOURCES