The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. We show that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. We identify this and another lysine residue as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, we identify cancer-associated mutations of PTEN that target its posttranslational modification and demonstrate how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN.

AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptations under the ATP-deprived conditions such as hypoxia. In the present study, we considered a role of AMPK in the adaptive response to hypoxia by examining whether AMPK is involved in the regulation of hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor that is critical for hypoxic induction of physiologically important genes. We demonstrate that hypoxia or CoCl 2 rapidly activated AMPK in DU145 human prostate cancer cells, and its activation preceded the induction of HIF-1␣ expression. Under these conditions, blockade of AMPK activity by a pharmacological or molecular approach significantly attenuated hypoxia-induced responses such as HIF-1 target gene expression, secretion of vascular endothelial growth factor, glucose uptake, and HIF-1-dependent reporter gene expression, indicating that AMPK is critical for the HIF-1 transcriptional activity and its target gene expression. Its functional requirement for HIF-1 activity was also demonstrated in several different cancer cell lines, but AMPK activation alone was not sufficient to stimulate the HIF-1 transcriptional activity. We further present data showing that AMPK transmits a positive signal for HIF-1 activity via a signaling pathway that is independent of phosphatidylinositol 3-kinase/AKT and several mitogen-activated protein kinases. Taken together, our results suggest that AMPK is a novel and critical component of HIF-1 regulation, implying its new roles in oxygen-regulated cellular phenomena.The energy status of the cell plays a crucial role for cell survival, and exposure of eukaryotic cells to metabolic stresses that accompany the depletion of intracellular ATP triggers specific and systemic adaptive responses. AMP-activated protein kinase (AMPK), 1 a heterotrimeric enzyme consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥), plays a critical role as an energy sensor in these responses (reviewed in Refs. 1-3). In response to nutritional or environmental stress factors that deplete intracellular ATP, AMPK is activated by allosteric binding of AMP (4, 5) and by phosphorylation by a still uncharacterized upstream AMPK kinase (6). Once activated, AMPK minimizes further ATP consumption by suppressing ATP-consuming anabolic pathways as well as activating ATP-generating catabolic pathways. The physiological or stress conditions known to activate AMPK include exercise (7-9), nutritional starvation (10), heat shock (11), oxidative stress (12), and ischemia/hypoxia (3, 13-15). Similar to the intracellular energy status, cellular oxygen concentration is precisely regulated in mammals to maintain cellular function and integrity. The reduced oxygen availability also initiates a series of adaptive responses, and many of these are mediated by HIF-1, which trans-activates several dozens of target genes whose protein products function to increase oxygen delivery and to enhance metabolic adaptation to anaerobic conditions (reviewed in Re...

Mutational alterations of the PTEN gene located on chromosome 10q23.3 have been frequently observed in a variety of human malignancies, including glioblastoma, melanoma, prostate cancer and endometrial cancer. 1-7 PTEN mutations and allelic deletions at 10q23 appear to be late events in glioblastoma, melanoma and prostate cancer, while in thyroid and endometrial cancers, PTEN alterations are found at an early stage, such as endometrial hyperplasia and benign thyroid tumors. 4 -9 Frequent germline or somatic mutations of PTEN have also been found in patients with Cowden disease and Bannayan-Zonana syndrome, which are autosomal dominant disorders characterized by the formation of multiple benign tumors and increased risk of malignant breast and thyroid tumors. 10,11 The PTEN gene encodes a protein product which shares high homology in its N-terminal region with the cytoskeletal protein tensin and the secretary vesicle protein auxilin. 1,2 The PTEN protein also contains a structural motif for a dual-specificity protein phosphatase. 12 PTEN acts as a phospholipid phosphatase, dephosphorylating PIP 3 with specificity for the phosphate group at the D3 position of the inositol ring. 13 PIP 3 is a lipid second messenger produced by PI3-kinase and activates a variety of signaling effectors such as AKT kinase. The lipid phosphatase activity of PTEN is essential for its ability to inhibit tumorigenesis and growth inhibition. 14,15 In human tumor cells lacking wild-type PTEN or in PTEN-deficient mice, PIP 3 levels are increased, leading to enhanced phosphorylation and activation of the survivalpromoting factor AKT kinase, indicating that PTEN exerts its tumor-suppressor function by negatively regulating the antiapoptotic PI3-kinase/AKT signaling pathway. 16 In addition, in immortalized PTEN-deficient mouse embryonic fibroblasts, PTEN restored apoptosis induced by stimuli such as UV irradiation. 17 The role of PTEN as a tumor-suppressor has also been attributed to its ability to modulate cell-cycle progression and cell motility. Expression of wild-type PTEN in PTEN-null glioblastoma or renal cell carcinoma cells causes cell-cycle arrest in the G 1 phase, inhibits colony formation and suppresses tumorigenicity in nude mice. 18 Exogenous expression of PTEN in fibroblasts and a glioma cell line with mutant PTEN alleles also suppresses cell migration, integrin-mediated cell spreading and focal adhesion. 19

During tumour progression, cancer cells use diverse mechanisms to escape from apoptosis-inducing stimuli, which may include receptor internalization, inhibition of signal pathways, and regulation of specific sets of genes. Substantial numbers of colon cancer cells have been observed to express Fas/Fas ligand, but are resistant to Fas-mediated apoptosis, suggesting that colonic tumours might develop specific mechanisms to overcome Fas-mediated apoptosis. Recently, cellular FLICE-like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas- or other receptor-mediated apoptosis and its altered high expression has a suspected association with tumour development or progression. In an effort to investigate the prevalence of cFLIP(L) alterations in colon carcinomas and their possible implications for the progression of colon cancers, cFLIP(L) expression was analysed in adenocarcinomas and adenomatous polyps of colon, with matched normal tissues, at RNA and protein levels, by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. cFLIP(L) transcripts were constitutively expressed in colon cancers and expression levels were significantly higher in carcinomas than in normal tissues (p<0.05). Overexpression of cFLIP(L) protein was found exclusively in carcinoma cells in all matched sets analysed and approximately three-fold induction was detected in cancer cells (p<0.05). The expression of cFLIP(L) protein was not significantly altered in adenomatous polyps compared with normal tissues. Taken together, these results strongly suggest that abnormal overexpression of cFLIP(L) is a frequent event in colon carcinomas and might contribute to in vivo tumour transformation.

Caveolin-1 (CAV1) acts as a growth suppressor in various human malignancies, but its expression is elevated in many advanced cancers, suggesting the oncogenic switch of its role during tumor progression. To understand the molecular basis for the growth-promoting function of CAV1, we characterized its expression status, differential roles for tumor growth, and effect on glucose metabolism in colorectal cancers. Abnormal elevation of CAV1 was detected in a substantial fraction of primary tumors and cell lines and tightly correlated with promoter CpG sites hypomethylation. Depletion of elevated CAV1 led to AMPK activation followed by a p53-dependent G 1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to ATP generation. Furthermore, CAV1 depletion downregulated glucose uptake, lactate accumulation, and intracellular ATP level, supporting that aerobic glycolysis is enhanced by CAV1. Consistently, CAV1 was shown to stimulate GLUT3 transcription via an HMGA1-binding site within the GLUT3 promoter. HMGA1 was found to interact with and activate the GLUT3 promoter and CAV1 increased the HMGA1 activity by enhancing its nuclear localization. Ectopic expression of HMGA1 increased glucose uptake, whereas its knockdown caused AMPK activation. In addition, GLUT3 expression was strongly induced by cotransfection of CAV1 and HMGA1, and its overexpression was observed predominantly in tumors harboring high levels of CAV1 and HMGA1. Together, these data show that elevated CAV1 upregulates glucose uptake and ATP production through HMGA1-mediated GLUT3 transcription, suggesting that CAV1 may render tumor cells growth advantages by enhancing aerobic glycolysis. Cancer Res; 72(16); 4097-109. Ó2012 AACR.