George Poulogiannis

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses

The glycolytic metabolism of cancers differs from normal tissues, allowing tumor cells to survive under oxidative stress. Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.

Phosphoproteomic Analysis Identifies Grb10 as an mTORC1 Substrate That Negatively Regulates Insulin Signaling

A search for substrates of a growth-promoting kinase revealed a regulatory feedback loop involved in tumor suppression. The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor–bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal–regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

The mTORC1 Pathway Stimulates Glutamine Metabolism and Cell Proliferation by Repressing SIRT4

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

PARK2 deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in Apc mutant mice

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25–27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% ∼0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli (APC) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with ApcMin mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome.

Poulogiannis G, Frayling I M & Arends M J
(2010) Histopathology56, 167–179

Metabolic stress controls mTORC1 lysosomal localization and dimerization by regulating the TTT-RUVBL1/2 complex.

The metabolism of glucose and glutamine, primary carbon sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that the ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function-all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cells metabolic state, directly regulating the functional assembly of mTORC1 and indirectly controlling the nutrient signal from Rags to mTORC1.

A constitutively activated form of the p110β isoform of PI3-kinase induces prostatic intraepithelial neoplasia in mice

Recent work has shown that ablation of p110β, but not p110α, markedly impairs tumorigenesis driven by loss of phosphatase and tensin homolog (PTEN) in the mouse prostate. Other laboratories have reported complementary data in human prostate tumor lines, suggesting that p110β activation is necessary for tumorigenesis driven by PTEN loss. Given the multiple functions of PTEN, we wondered if p110β activation also is sufficient for tumorigenesis. Here, we report that transgenic expression of a constitutively activated p110β allele in the prostate drives prostate intraepithelial neoplasia formation. The resulting lesions are similar to, but are clearly distinct from, the ones arising from PTEN loss or Akt activation. Array analyses of transcription in multiple murine prostate tumor models featuring PI3K/AKT pathway activation allowed construction of a pathway signature that may be useful in predicting the prognosis of human prostate tumors.

Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

K‐ras mutations are found in 40–50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K‐ras mouse model (K‐rasAsp12/Cre), with transient intestinal Cre activation by β‐Naphthoflavone (β‐NF) treatment, displayed transgene recombination and K‐rasAsp12 expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with ApcMin/+ mice, the K‐rasAsp12/Cre/ApcMin/+ offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 ± 5.4 weeks from 20.9 ± 4.7 weeks (control ApcMin/+ mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5‐fold and 5.7‐fold, respectively, in K‐rasAsp12/Cre/ApcMin/+ mice compared with ApcMin/+ mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K‐rasAsp12 and ApcMin co‐operation, the Mitogen‐activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K‐rasAsp12 increased activation of Mapk and Akt signalling pathway targets phospho‐extracellular signal‐regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo‐oxygenase 2 (Cox2) and T‐cell lymphoma invasion and metastasis 1 (Tiam1) in K‐rasAsp12/Cre/ApcMin/+ adenomas compared with that of ApcMin/+ adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator‐activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c‐myc remained unchanged. In conclusion, intestinal expression of K‐rasAsp12 promotes mutant Apc‐initiated intestinal adenoma formation in vivo more in the large intestine than the small intestine, with evidence of synergistic co‐operation between mutant K‐ras and Apc involving increased expression of some Wnt‐pathway target genes.

Prognostic relevance of DNA copy number changes in colorectal cancer.

In a study of 109 colorectal cancers, DNA copy number aberrations were identified by comparative genomic hybridization using a DNA microarray covering the entire genome at an average interval of less than 1 Mbase. Four patterns were revealed by unsupervised clustering analysis, one of them associated with significantly better prognosis than the others. This group contained tumours with short, dispersed, and relatively few regions of copy number gain or loss. The good prognosis of this group was not attributable to the presence of tumours showing microsatellite instability (MSI‐H). Supervised methods were employed to determine those genomic regions where copy number alterations correlate significantly with multiple indices of aggressive growth (lymphatic spread, recurrence, and early death). Multivariate analysis identified DNA copy number loss at 18q12.2, harbouring a single gene, BRUNOL4 that encodes the Bruno‐like 4 splicing factor, as an independent prognostic indicator. The data show that the different patterns of DNA copy number alterations in primary tumours reveal prognostic information and can aid identification of novel prognosis‐associated genes. Copyright

Conditional expression of mutated K-ras accelerates intestinal tumorigenesis in Msh2-deficient mice

K-ras mutation occurs in 40–50% of human colorectal adenomas and carcinomas, but its contribution to intestinal tumorigenesis in vivo is unclear. We developed K-rasV12 transgenic mice that were crossed with Ah-Cre mice to generate K-rasV12/Cre mice, which showed β-naphthoflavone-induction of Cre-mediated LoxP recombination that activated intestinal expression of K-rasV12 4A and 4B transcripts and proteins. Only very occasional intestinal adenomas were observed in β-naphthoflavone-treated K-rasV12/Cre mice aged up to 2 years, suggesting that mutated K-ras expression alone does not significantly initiate intestinal tumourigenesis. To investigate the effects of mutated K-ras on DNA mismatch repair (MMR)-deficient intestinal tumour formation, these mice were crossed with Msh2−/− mice to generate K-rasV12/Cre/Msh2−/− offspring. After β-naphthoflavone treatment, K-rasV12/Cre/Msh2−/− mice showed reduced average lifespan of 17.3±5.0 weeks from 26.9±6.8 (control Msh2−/− mice) (P<0.01). They demonstrated increased adenomas in the small intestine from 1.41 (Msh2−/− controls) to 7.75 per mouse (increased fivefold, P<0.01). In the large intestine, very few adenomas were found in Msh2−/− mice (0.13 per mouse) whereas K-rasV12/Cre/Msh2−/− mice produced 2.70 adenomas per mouse (increased 20-fold, P<0.01). Over 80% adenomas from K-rasV12/Cre/Msh2−/− mice showed transgene recombination with expression of K-rasV12 4A and 4B transcripts and proteins. Sequencing of endogenous murine K-ras showed mutations in two out of 10 tumours examined from Msh2−/− mice, but no mutations in 17 tumours from K-rasV12/Cre/Msh2−/− mice. Expression of K-rasV12 in tumours caused activation of the mitogen-activated protein kinase and Akt/protein kinase B signaling pathways, demonstrated by phosphorylation of p44MAPK, Akt and GSK3β, as well as transcriptional upregulation of Pem, Tcl-1 and Trap1a genes (known targets of K-rasV12 expression in stem cells). Thus, mutated K-ras cooperates synergistically with MMR deficiency to accelerate intestinal tumorigenesis, particularly in the large intestine.