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Creative activism is an approach to education that asks: 'What can happen when we take learning outside the classroom and think of it happening everywhere?' Two charities – House of Imagination and Cambridge Curiosity and Imagination – have been asking this question in their creative place-making programmes working with socially engaged artists and communities linked to primary schools in Bath and Cambridge. Young children and adults co-create and speculate about the future of their communities and environments in these different geographical locations. This article draws together the authors' shared understanding of creative pedagogies and the value to everyone of working in this way.

Background: Activation of signaling pathways, loss of tumor suppressors and oncogenic mutations often lead to dysregulation of glycolysis and tumorigenesis. We performed expression analysis on genetically-defined human isogenic breast cancer cell lines and with PI3K inhibitor exposure to determine impact on a patient-relevant Metastasis Score (MS) (proliferation index) and an assembled “glycolytic index”. A better understanding of the links between cellular metabolism and proliferation may yield insights into tumor cell biology and shed light on potential companion diagnostic and therapeutic opportunities. Methods: A parental MCF10A and three isogenic cell lines harboring K-ras (G12V), PI3Kα (H1047R), and p53 null were grown overnight in the presence of high levels of growth factors in tissue culture flasks. The parental and PI3Kα cells were treated with 0.3 µM, 1 µM,3 µM GDC-0941 or DMSO. All cell lines were incubated for an additional 24 hr. RNA was extracted with RNeasy (Qiagen) and expression analysis performed by RT-PCR. A total of 28 genes were profiled that included 14 genes in a metastasis score (MS) (Tutt et al), 11 glycolysis-associated genes and 3 reference genes. Expression of each gene was calculated using the ΔΔCT method and “summed” with equal weighting to yield an MS and a “glycolytic index”. Results: The MS and phenotypes for the four untreated cell lines were similar under the monolayer culture conditions used. The parental and PI3Kα cells had a similar “glycolytic index” while p53 null and K-ras cells had elevated indices. Treatment of the parental and PI3Kα with GDC-0941, a PI3K inhibitor, showed a dose-dependent decrease in the expression of constituent MS genes and the glycolytic genes as well as the combined scores. The correlation coefficient between MS and the glycolytic index was 0.77. The four genes in MS with the greatest dose-dependent decrease in expression (BUB1, CCNB1, MYBL2 and UBE2S) are associated with CDK1, a seminal participant in cell cycle regulation. The two genes from the “glycolytic index” with the greatest decrease, HK and PKM, are directly involved in glycolysis. The effect of GDC-0941, as reflected by MS and the “glycolytic index” was more pronounced in PI3Kα than the parental cells (p-value=0.0005). Conclusion: The good correlation observed between the MS and “glycolytic index “in the treated cell lines supports an association between proliferation and metabolism upon restriction of cell signal transduction. The dose-dependent expression decreases in the two indices and their constituent genes, suggest that this PI3K inhibitor successfully perturbs both fundamental processes of the cell. Given the ultimate targeting of proliferation suppression, these scores may serve as useful tools in evaluating therapeutic agents, independent of the specific intended pathways. The similar proliferation levels observed in untreated MCF10A and three mutated cell lines suggest either that single oncogenic mutations are insufficient to affect MS or that the in vitro conditions are masking the metastatic/invasive phenotype. Studies are underway to evaluate cells grown in 3D Matrigel that more accurately mimic the tumor microenvironment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-19.

Background: We previously reported that a published “Metastasis Score” (MS) could be used to evaluate the effects of a PI3K inhibitor (GDC-0941) on mutated human isogenic breast cancer cell lines. MS, based on the expression of14 genes, has been shown to predict distant metastasis in ER(+), node (−), breast cancer. We were interested to determine the impact of gefitinib (EGFR inhibitor) and PLX-4720 (selective inhibitor of BRAF V600E) on MS to assess the applicability of this score to a broader class of targeted agents. In addition, given the cross-talk between metabolism and proliferation, we also profiled genes involved in glycolysis, fatty acid metabolism and oxidative phosphorylation. Methods: Parental MCF10A (WT for all genes) and isogenic lines of MCF10A harboring PI3K (H1047R), p53 null or KRAS(G12V) mutations were cultured overnight in DMEM:F12 media under identical conditions. Cells were then treated with gefitinib, PLX4720, or DMSO (vehicle control) and further incubated for 24hr. Expression analysis was performed by RT-PCR. Results: The MS of the PI3K (H1047R) and p53 null lines was higher than the parental line and lower for the KRAS (G12V) line. Treatment of parental, PI3K and KRAS cell lines with gefitinib resulted in dose-dependent decreases in MS, as reported for GDC-0941, with a higher dose required to inhibit growth of PI3K (H1047R) cells. Treatment of p53 null cells with gefitinib, however, had only a modest effect on MS. In contrast to gefitinib, MS increased with PLX-4720 treatment in all 4 lines; the greatest increase was observed in KRAS (G12V) cells. The expression of metabolic genes differed significantly depending on the oncogenic mutation harbored by the cell line. ACTA2, ACLY, RPIA, KHK, GLS2 were most highly expressed in p53 null but lowest in KRAS (G12V), while GLUT5, PFKFB4, ENO3, SLC27A1, PGK2, GLUT1, TKT were expressed most highly in KRAS (G12V) but lowest in PI3K(H1047R). Upon treatment with gefitinib or PLX-4720, genes that were downregulated in the mutated lines generally showed a dose-dependent increase; those that were upregulated relative to the parental, showed a dose-dependent decrease with treatment. Discussion: The decrease in MS of the parental, PI3K (H1047R) and KRAS (G12V) cell lines treated with gefitinib supports the impact of this EGFR inhibitor on cell proliferation. The modest effect on MS in p53 null cells supports the findings that sensitivity to gefitinib requires active p53 in order to induce apoptosis through a p53-dependent pathway. Increases in MS were observed in all 4 cell lines treated with PLX-4720, with the largest increase observed in KRAS (G12V). The enhanced proliferation of a BRAF WT cell line with a KRAS mutation is consistent with the Ras-dependent nature of this pathway. The metabolic genes showed diverse expression patterns that differed with different oncogenic mutations and likely reflect the multiple mechanisms controlling metabolism in cancer. An improved understanding of the expression of metabolic genes relative to proliferation in cell lines with various oncogenic mutations may provide additional insights into the dysregulation of these cellular processes and the possible role of anti-metabolite intervention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-04.

Targeting tumor metabolism is becoming a major new area of pharmaceutical endeavor. Consequently, a systematic search to define whether there are specific energy source dependencies in tumors, and how these might be dictated by upstream driving genetic mutations, is now required. This is particularly relevant given the potential ability of cancer cells to switch between metabolic pathways. The PI3K-Akt-mTOR signaling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival. This pathway has also been associated with metabolic dysregulation, particularly gluconeogenesis, largely through effects on Akt and mTOR. We have used isogenic cell models, wherein homologous recombination is used to specifically delete endogenous mutant PI3KCA alleles or introduce PIK3CA activating mutations in human cell lines. By performing rtPCR on candidate metabolic genes, we have found gene expression signatures indicative of a consistent up-regulation of glycolysis in PIK3CA mutant cells. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. In MCF10A breast epithelial ‘knock-in’ lines, hexokinase 2 (HK2) is the primary target of up-regulation. However, in HCT116 colon cancer cells that are heterozygous for the H1047R PIK3CA mutation, knocking-out mutant PIK3CA resulted in a marked reduction of Glut1. Altered expression of asparagine synthetase (ASNS) and glutaminase (GLS2) was also observed in both models, suggestive of a potential increased dependency on glutaminolysis, while up-regulation of the fatty acid transporter SLC27A2 was observed in HCT116 PIK3CA mutant cells. To determine whether any of these metabolic expression changes impart hard-wired nutrient dependencies, we next performed nutrient switching experiments. These demonstrated that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients such as alternative monosaccharides, fatty acids or aspartic acid. The extent of glucose dependency of the PIK3CA mutant cells suggested that targeted disruption of the glycolytic pathway should inhibit growth, even in the presence of glucose. The use of hexokinase 2 siRNA to disrupt glycolysis did indeed result in anti-proliferative effects that were selective for the PIK3CA mutant cells. In conclusion, we have demonstrated that glycolysis-associated genes are up-regulated in PIK3CA mutant cells and that the shift to a glycolytic phenotype does not appear to be controlled by a single rate limiting molecule. Despite suggestions that cancer cells are adept at utilizing alternative nutrient sources, this study has demonstrated that PIK3CA mutant cell are not able to compensate for glucose withdrawal. This specific dependence on glucose for growth, identified using precise isogenic models, was also present across a wider panel of cancer cell lines harboring endogenous PIK3CA mutations. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B140.

Background: During tumorigenesis, cells acquire mutations that confer selective advantages for growth. These mutations may reprogram cellular metabolism to meet the requirements of rapid proliferation and survival in an environment of fluctuating and scarce nutrients. Phosphoinositide-3-kinases (PI3Ks) play key roles in fundamental cellular processes and are frequently mutated in a broad range of cancers. The aim of this study was to investigate how a single endogenous activating mutation in PI3K, H1047R, affects the metabolic programming of human breast epithelial cells. Methods: We utilized X-MAN isogenic cells derived from MCF10a mammary epithelial cells, which harbour a single H1047R PIK3CA mutation introduced via AAV-mediated homologous recombination and compared them to matched parental cells with a wild type (WT) allele. This system allows use of endogenous promoters thereby more accurately recapitulating the genomic architecture in human tumours. Metabolic gene expression profiles were measured by RT-PCR. Results: We identified a multi-gene expression signature indicative of increased dependency on the glycolytic pathway. Hexokinase 2, which is involved at an early stage of the glycolytic pathway, was strongly up-regulated along with the lactate transporters MCT1 and 4. The fructose transporter GLUT5 was nearly undetectable in H1047R PIK3CA cells, suggesting a strong preference for glucose and elevated levels of aerobic glycolysis. PI3K mutant cells also down-regulated genes in the pentose phosphate pathway-shunt, tricarboxylic acid cycle and glutaminolysis pathways, indicating that aerobic glycolysis is heavily favoured. Treatment with the PI3K inhibitor GDC-0941 led to a general reversal of gene expression for these pathways, confirming the PI3K specificity of these metabolic alterations. We proceeded to investigate growth dependence of the H1047R PIK3CA and parental MCF10a cells on various nutrient sources (e.g. glucose, fructose, galactose and glutamine). While the growth rates of cells WT for PIK3CA were not significantly affected by glucose withdrawal and nutrient replacement, growth of mutant PIK3CA cells was dramatically compromised in the absence of glucose, revealing a specific dependency for this nutrient as an energy substrate. Consistent with PI3K activation mediating glucose dependency, treatment of H1047R PIK3CA cells with GDC-0941 promoted growth of the cells under low or no glucose conditions. Conclusion: Isogenic cells WT for PIK3CA are capable of utilizing a range of nutrient sources, while cells harboring H1047R PIK3CA appear hard-wired to exclusively utilize glucose. These data suggest that targeting glucose transporters or glycolytic enzymes may be a powerful therapeutic strategy for mutant PI3K-cancers. The overt glucose dependence of cells with this mutation may also offer routes to non-invasively image PI3K-pathway activated tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1274. doi:10.1158/1538-7445.AM2011-1274

Background: Human derived cell lines represent an important model system for fundamental research and translational medicine. We sought to gather patient-relevant information by examining a previously published metastasis score (MS) (proliferation index) for operable breast cancer in well-characterized isogenic cell lines. We chose to examine the impact on MS of specific genetic alterations in known oncogenes and consequence of treatment with a class I PI3K inhibitor. Investigation of the proliferation index of cells without the complexity of cell mixtures found in tumor microenvironment may shed light on tumor cell-specific transcriptional dysregulation. Methods: Nontumorigenic MCF10A and isogenic cell lines harboring (G12V), PI3Kα (H1047R) and p53 (null) mutations were cultured in T75cm tissue culture flasks overnight. MCF10A and isogenic PI3Kα (H1047R) cell lines were seeded and allowed to adhere overnight before addition of 0.3 µM, 1 µM and 3 µM GDC-0941 or DMSO control and incubated for 24 hours. Total RNA was prepared using RNeasy Kit (Qiagen) from cells. Expression profiling was carried out using RT-PCR for the previously described metastasis score (Tutt et al, 2009) that includes 14 genes and 3 reference genes. Results: MCF10A and the isogenic mutant cell lines had equivalent levels of proliferation as measured by MS and similar phenotypes under these monolayer culture conditions with high levels of growth factors. Modest dose-dependent decreases in the expression level of the constituent MS genes (1.5-3 fold) were observed with GDC-0191 exposure that translated into a marked decrease in overall MS (14 to 4) due to their equally weighted additive impact. The four genes that demonstrated the greatest decrease were CCNB1, BUB1, MYBL2, and UBE2S. These genes are highly associated with cyclin-dependent kinase (CDK1), a key player in cell cycle regulation. The MS score for these isogenic lines are also being defined under in vitro assay conditions that more accurately mimic the tumor microenvironment; in particular a 3-dimentional Matrigel assay that unleashes a marked differential metastatic/invasive phenotype in mutant vs. WT cells. Conclusion: MCF10A and isogenic cell lines harboring KRAS (G12V), PI3Kα (H1047R) and p53 (null) mutations had equivalent levels of proliferation as measured by MS. These results suggest that a combination rather than a single oncogenic mutant are required to alter the MS, although in vitro conditions likely play an important role in fully unmasking genotype-specific phenotypes in these clean gain-of-function models. Exposure of parental and PI3K (H1047R) cell lines to PI3K inhibitor GDC-0941 led to a dose-dependent decrease in constituent genes and composite MS. These results suggest that even though genes in the PI3K/PTEN pathway are not included in the molecular score, perturbation of this signaling pathway leads to alteration of the MS, by affecting cell cycle progression. Future studies are required to determine if alteration of MS in cell lines treated with different targeted oncogenic pathway inhibitors will recapitulate patient tumor response. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A33.