AMP-Activated Protein Kinase:Friend or Foe in Cancer

The AMP-activated protein kinase (AMPK) is activated by energy stress and restores homeostasis by switching on catabolism, while switching off cell growth and proliferation. Findings that AMPK acts downstream of the tumor suppressor LKB1 have suggested that AMPK might also suppress tu-morigenesis. In mouse models of B and T cell lymphoma in which genetic loss of AMPK occurred before tumor initiation, tumorigenesis was accelerated , confirming that AMPK has tumor-suppressor functions. However, when loss of AMPK in a T cell lymphoma model occurred after tumor initiation , or simultaneously with tumor initiation in a lung cancer model, the disease was ameliorated. Thus, once tumorigenesis has occurred, AMPK switches from tumor suppression to tumor promotion. Analysis of alterations in AMPK genes in human cancers suggests similar dichotomies, with some genes being frequently amplified while others are mutated. Overall, while AMPK-activating drugs might be effective in preventing cancer, in some cases AMPK inhibitors might be required to treat it

Involvement of PPARy in the antitumoral action of cannabinoids on hepatocellular carcinoma

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In\ud this study, we examined whether the PPARc-activated pathway contributed to the antitumor effect of two cannabinoids,\ud D9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity\ud and intracellular level of PPARc mRNA and protein, which was abolished by the PPARc inhibitor GW9662. Moreover, genetic\ud ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARc decreased the cannabinoid-induced\ud cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC\ud tumors in mice. In addition, PPARc knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62,\ud suggesting that PPARc is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the\ud endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARc expression and induced p62\ud accumulation, which was counteracted by overexpression of PPARc in TRIB3-knocked down cells. Taken together, we\ud demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo,\ud are modulated by upregulation of PPARc-dependent pathways

Involvement of PPARy in the antitumoral action of cannabinoids on hepatocellular carcinoma

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARc-activated pathway contributed to the antitumor effect of two cannabinoids, D9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARc mRNA and protein, which was abolished by the PPARc inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARc decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARc knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARc is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARc expression and induced p62 accumulation, which was counteracted by overexpression of PPARc in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARc-dependent pathways

Androgen Deprivation Induces Reprogramming of Prostate Cancer Cells to Stem-Like Cells

In the past few years, cell plasticity has emerged as a mode of targeted therapy evasion in prostate adenocarcinoma. When exposed to anticancer therapies, tumor cells may switch into a different histological subtype, such as the neuroendocrine phenotype which is associated with treatment failure and a poor prognosis. In this study, we demonstrated that long-term androgen signal depletion of prostate LNCaP cells induced a neuroendocrine phenotype followed by re-differentiation towards a ?stem-like? state. LNCaP cells incubated for 30 days in charcoal-stripped medium or with the androgen receptor antagonist 2-hydroxyflutamide developed neuroendocrine morphology and increased the expression of the neuroendocrine markers ?III-tubulin and neuron specific enolase (NSE). When cells were incubated for 90 days in androgen-depleted medium, they grew as floating spheres and had enhanced expression of the stem cell markers CD133, ALDH1A1, and the transporter ABCB1A. Additionally, the pluripotent transcription factors Nanog and Oct4 and the angiogenic factor VEGF were up-regulated while the expression of E-cadherin was inhibited. Cell viability revealed that those cells were resistant to docetaxel and 2-hidroxyflutamide. Mechanistically, androgen depletion induced the decrease in AMP-activated kinase (AMPK) expression and activation and stabilization of the hypoxia-inducible factor HIF-1?. Overexpression of AMPK in the stem-like cells decreased the expression of stem markers as well as that of HIF-1? and VEGF while it restored the levels of E-cadherin and PGC-1?. Most importantly, docetaxel sensitivity was restored in stem-like AMPK-transfected cells. Our model provides a new regulatory mechanism of prostate cancer plasticity through AMPK that is worth exploring.Fundación Tatiana Pérez de Guzmán el Buen

Genotoxic Damage Activates the AMPK-α1 Isoform in the Nucleus via Ca2+/CaMKK2 Signaling to Enhance Tumor Cell Survival

2017 American Association for Cancer Research. Many genotoxic cancer treatments activate AMP-activated protein kinase (AMPK), but the mechanisms of AMPK activation in response to DNA damage, and its downstream consequences, have been unclear. In this study, etoposide activates the a1 but not the a2 isoform of AMPK, primarily within the nucleus. AMPK activation is independent of ataxia-telangiectasia mutated (ATM), a DNA damage-activated kinase, and the principal upstream kinase for AMPK, LKB1, but correlates with increased nuclear Ca2þ and requires the Ca2þ/calmodulin-dependent kinase, CaMKK2. Intriguingly, Ca2þ-dependent activation of AMPK in two different LKB1-null cancer cell lines caused G1-phase cell-cycle arrest, and enhanced cell viability/ survival after etoposide treatment, with both effects being abolished by knockout of AMPK-a1 and a2. The CDK4/6 inhibitor palbociclib also caused G1 arrest in G361 but not HeLa cells and, consistent with this, enhanced cell survival after etoposide treatment only in G361 cells. These results suggest that AMPK activation protects cells against etoposide by limiting entry into S-phase, where cells would be more vulnerable to genotoxic stress. Implications: These results reveal that the a1 isoform of AMPK promotes tumorigenesis by protecting cells against genotoxic stress, which may explain findings that the gene encoding AMPK-a1 (but not -a2) is amplified in some human cancers. Furthermore, a1-selective inhibitors might enhance the anticancer effects of genotoxic-based therapies

Targeting AMP-activated kinase impacts hepatocellular cancer stem cells induced by long-term treatment with sorafenib

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. HCC treatment is hindered by the frequent emergence of chemoresistance to the multikinase inhibitor sorafenib, which has been related to the presence of cancer stem cells (CSCs) that self‐renew and often escape therapy. The key metabolic sensor AMP‐activated kinase (AMPK) has recently been recognized as a tumour growth regulator. In this study, we aimed to elucidate the role of AMPK in the development of a stem cell phenotype in HCC cells. To this end, we enriched the CSC population in HCC cell lines that showed increased expression of drug resistance (ALDH1A1, ABCB1A) and stem cell (CD133, Nanog, Oct4, alpha fetoprotein) markers and demonstrated their stemness phenotype. These cells were refractory to sorafenib‐induced cell death. We report that sorafenib‐resistant cells had lower levels of total and phosphorylated AMPK as well as its downstream substrate, ACC, compared with the parental cells. Interestingly, AMPK knockdown with siRNA or inhibition with dorsomorphin increased the expression of stem cell markers in parental cells and blocked sorafenib‐induced cell death. Conversely, the upregulation of AMPK, either by transfection or by pharmacological activation with A‐769662, decreased the expression of ALDH1A1, ABCB1A, CD133, Nanog, Oct4, and alpha fetoprotein, and restored sensitivity to sorafenib. Analysis of the underlying mechanism points to hypoxia‐inducible factor HIF‐1α as a regulator of stemness. In vivo studies in a xenograft mouse model demonstrated that stem‐like cells have greater tumourigenic capacity. AMPK activation reduced xenograft tumour growth and decreased the expression of stem cell markers. Taken together, these results indicate that AMPK may serve as a novel target to overcome chemoresistance in HCC

AMPK Causes Cell Cycle Arrest in LKB1-deficient Cells via Activation of CAMKK2

The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca(2+)/calmodulin-dependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1-AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 re-expression in these cells causes cell cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca(2+) ionophore A23187 caused a G1-arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, CAMKK2 mutant also caused G1-arrest similar to that caused by expression of LKB1, while expression of a dominant negative AMPK mutant, or a double knockout of both AMPK-α subunits, also prevented the cell cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell cycle arrest can be restored by re-expressing LKB1 or a constitutively active CAMKK2, or by pharmacological agents that increase intracellular Ca(2+) and thus activate endogenous CAMKK2. IMPLICATIONS: Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented

The cannabinoid WIN 55,212-2 prevents neuroendocrine differentiation of LNCaP prostate cancer cells

BACKGROUND: Neuroendocrine (NE) differentiation represents a common feature of prostate cancer and is associated with accelerated disease progression and poor clinical outcome. Nowadays, there is no treatment for this aggressive form of prostate cancer. The aim of this study was to determine the influence of the cannabinoid WIN 55,212-2 (WIN, a non-selective cannabinoid CB1 and CB2 receptor agonist) on the NE differentiation of prostate cancer cells. METHODS: NE differentiation of prostate cancer LNCaP cells was induced by serum deprivation or by incubation with interleukin-6, for 6 days. Levels of NE markers and signaling proteins were determined by western blotting. Levels of cannabinoid receptors were determined by quantitative PCR. The involvement of signaling cascades was investigated by pharmacological inhibition and small interfering RNA. RESULTS: The differentiated LNCaP cells exhibited neurite outgrowth, and increased the expression of the typical NE markers neuron-specific enolase and ?III tubulin (?III Tub). Treatment with 3??M WIN inhibited NK differentiation of LNCaP cells. The cannabinoid WIN downregulated the PI3K/Akt/mTOR signaling pathway, resulting in NE differentiation inhibition. In addition, an activation of AMP-activated protein kinase (AMPK) was observed in WIN-treated cells, which correlated with a decrease in the NE markers expression. Our results also show that during NE differentiation the expression of cannabinoid receptors CB1 and CB2 dramatically decreases. CONCLUSIONS: Taken together, we demonstrate that PI3K/Akt/AMPK might be an important axis modulating NE differentiation of prostate cancer that is blocked by the cannabinoid WIN, pointing to a therapeutic potential of cannabinoids against NE prostate cancer.Ministerio de Economía y CompetitividadJunta de Comunidades de Castilla-La ManchaFundación Tatiana Pérez de Guzmán el BuenoFondo Europeo de Desarrollo RegionalComunidad de Madri

Phenformin, but not metformin, delays development of T-cell acute lymphoblastic leukemia/lymphoma via cell-autonomous AMPK activation

Summary: AMPK acts downstream of the tumor suppressor LKB1, yet its role in cancer has been controversial. AMPK is activated by biguanides, such as metformin and phenformin, and metformin use in diabetics has been associated with reduced cancer risk. However, whether this is mediated by cell-autonomous AMPK activation within tumor progenitor cells has been unclear. We report that T-cell-specific loss of AMPK-α1 caused accelerated growth of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by PTEN loss in thymic T cell progenitors. Oral administration of phenformin, but not metformin, delayed onset and growth of lymphomas, but only when T cells expressed AMPK-α1. This differential effect of biguanides correlated with detection of phenformin, but not metformin, in thymus. Phenformin also enhanced apoptosis in T-ALL cells both in vivo and in vitro. Thus, AMPK-α1 can be a cell-autonomous tumor suppressor in the context of T-ALL, and phenformin may have potential for the prevention of some cancers. : The roles of AMPK in cancer and of biguanides in its prevention or treatment are controversial. Vara-Ciruelos et al. now report that genetic loss of AMPK in T cells accelerates T cell acute lymphoblastic leukemia/lymphoma, whereas the biguanide phenformin, but not metformin, protects against its development in a cell-autonomous, AMPK-dependent manner. Keywords: AMP-activated protein kinase, AMPK, biguanides, metformin, phenformin, T-ALL, T cell acute lymphoblastic leukemia/lymphom

Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and ?III tubulin (?III tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1?M AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer