Search

Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We now link cancer-specific transcription factor (TF) expression dysregulation to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their pre-malignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF-expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMP), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). CIMP/CEMP coordination appears to be driven by augmented downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The dysregulated expression of TFs related to CIMP/CEMP subtypes occurs early during CRC development, detectable in pre-malignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes. Citation Format: Yuba R. Bhandari, Vinod Krishna, Rachael Powers, Sehej Parmar, Sara-Jayne Thursby, Ekta Gupta, Ozlem Kulak, Prashanth Gokare, Joke Reumers, Liesbeth Van Wesenbeeck, Kurtis E. Bachman, Stephen B. Baylin, Hariharan Easwaran. Transcription factor expression repertoire basis for epigenetic and transcriptional subtypes of colorectal cancers. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A012.

Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNγ+ T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of 5AZA-C + DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. Significance: Combined epigenetic and polyamine-reducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.

Colorectal cancer (CRC) is classified into distinct subtypes based on genetic, epigenetic and/or transcriptional subtypes. The underlying mechanisms that drive these subtypes are unknown. By integrating whole genome DNA methylation, gene expression, chromatin patterns, and enhancer elements in CRCs, we show that expression of large numbers of transcriptional factors (TFs) is deregulated in CRCs and these expression patterns drive epigenetic and transcriptional subtypes of CRCs. The first key result emerging is a new understanding of important DNA methylation patterns central to classifying CRCs. Of these, different degrees of cancer associated promoter methylation have been recognized that suppress expression, and/or inducibility of important tumor suppressor genes. Stratified by high to low degrees of hypermethylation, this pattern is termed CpG-island promoter DNA methylation phenotypes or CIMP. We now tightly link CIMP to a cancer-specific hypermethylation at enhancer regulatory elements we term, CpG- enhancer methylation phenotype (CEMP). CIMP and CEMP are driven by decreased expression of multiple TFs with binding sites in these regulatory elements, and the involved TFs are frequently identical between the two regions. In contrast, a different set of TF factors are downregulated in non-CIMP tumors, again in conjunction with methylation patterns linking promoter and enhancer target sites for the involved TFs. Further, enhancer regulatory elements that are hypomethylated in cancer relative to normal tissue are associated with increased expression of another distinct set of TFs with target sites in the corresponding enhancers. Importantly, the TF expression patterns, and the above CIMP, CEMP, and hypomethylation patterns, are established early in the pre-malignant adenomas. Importantly, pan-cancer analyses showed that the above CIMP and CEMP subtypes occur across important cancer types in addition to CRC, again in parallel with marked downregulation of TFs that have target sites in the methylated regulatory elements. All of the above newly defined patterns also provide a basis for the transcriptome-based consensus molecular subtypes (CMS) of CRC. Overall, our studies may provide key understanding of how cancers, and their initiation and progression, are diseases which at the end of the day depend on abnormal patterns of TF expression, and how these relate to methylation alterations at target enhancer and promoter regions. Citation Format: Yuba Bhandari, Rachael Powers, Sehej Parmar, Vinod Krishna, Kurtis E. Bachman, Steve Baylin, Hariharan Easwaran. Transcription factor repertoire basis for epigenetic and consensus molecular subtypes of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2107.

Background: Familial adenomatous polyposis (FAP) is the most common hereditary polyposis syndrome. It is an autosomal dominant inherited disorder characterized by the early onset of hundreds to thousands of adenomatous polyps throughout the colon. If left untreated, nearly all individuals with this syndrome develop colorectal cancer (CRC) by the third decade of life. Prophylactic colectomy is the standard of care, but individuals remain at risk for malignant transformation of duodenal polyps, rectal polyps for those who have undergone rectal-sparing surgeries, and ileal pouch polyps for those with ileal pouch-anal anastomoses. Multiple studies with both nonselective and selective cyclooxygenase inhibitors (such as sulindac or celecoxib) have shown that anti-inflammatory agents may prevent the formation and inhibit the growth of colorectal adenomatous polyps. However, toxicities associated with these agents and their limited efficacy have prevented their further development. Therefore, there is a high unmet need for novel treatment options to reduce polyp burden, delay or eliminate the need for colectomy and recurrent rectal surgery, and intercept the development of adenocarcinomas in individuals with FAP. Polyps from individuals with FAP display inflammatory features associated with the activation of the IL-23/IL-17/JAK/STAT3 pathway. This inflammation is thought to contribute to further carcinogenesis, culminating in tumor development. Specifically, IL-23 is linked to tumor growth and progression in CRC, and adenomas with high-grade dysplasia showed elevated levels of IL-17A and pSTAT3. Guselkumab, a human monoclonal antibody directed against the p19 subunit of IL-23, specifically targets IL-23 and inhibits its interaction with the IL-23 receptor. Pre-clinical models suggest that inhibition of IL-23 signaling will result in less inflammation and reduce tumor development. Methods: This randomized, blind, placebo-controlled study will evaluate the safety and efficacy of guselkumab in adults with FAP (genetic or clinical diagnosis) who have already undergone colectomy. Polyps with a sum of diameters ≥10 mm in the rectum or pouch are required. Subjects will be randomized equally to one of three study arms: 100 mg, 300 mg, or placebo given subcutaneously every 4 weeks for 6 doses. The primary efficacy endpoint is percentage change from baseline in rectal/pouch polyp burden after 24 weeks. Secondary efficacy endpoints include duodenal polyp burden change and changes in InSiGHT and Spigelman staging. Exploratory translational research objectives will explore changes in RNA expression profiles, epigenomic profiles, cytokine levels, and the microbiome. Exclusion criteria include prior IL-23 targeted therapies and any polyps >1 cm that cannot be removed. While participating, subjects are required to stop any other FAP-directed drug therapy except for aspirin. As of January 2019, 18% of the planned 72 subjects have been enrolled. ClinicalTrials.gov Identifier: NCT03649971. Citation Format: Eduardo Vilar-Sanchez, Carol Burke, Marcia R. Cruz Correa, Evelien Dekker, William M. Grady, Philippe Grandval, Bryson W. Katona, Xavier Llor, Douglas L. Riegert-Johnson, Jean-Christophe Saurin, Peter Stanich, Daniel A. Sussman, David Weinberg, Edward F. Attiyeh, Devanand Joseph, Kelly Raybold, Gary V. Borzillo, Thomas J. Prior, Michael Smith, Hong Xie, Kurtis E. Bachman, Jeffrey R. Infante, Niloy Jewel Samadder. A phase 1b, multicenter, randomized, blinded, placebo-controlled study to evaluate the efficacy of guselkumab in subjects with familial adenomatous polyposis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT236.

In the letter by Haffner et al. (1), they report that seminoma cell-intrinsic DNA hypomethylation is associated with endogenous retroviral expression, an IFN response, and lymphocytic infiltration. Their data complement and support our recent therapeutic study in a mouse model of ovarian cancer (2) and support our observations that low doses of the DNA methyltransferase (DNMT) inhibitor azacytidine (AZA), in combination with the histone deacetylase inhibitor, givinostat, activate type 1 IFN signaling in ovarian (2) and lung (3) cancer cells to increase numbers and activation of immune cells in the tumor microenvironment and to increase sensitivity to the … [↵][1]5To whom correspondence may be addressed. Email: zahnoci{at}jhmi.edu or sbaylin{at}jhmi.edu. [1]: #xref-corresp-1-1

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

Lapatinib is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human EGFR-2 (HER2) tyrosine kinase domains. To explore the potential utility of lapatinib for the treatment of esophageal squamous cell carcinoma (ESCC), we examined the expression profiles of EGFR and HER2 in tumor tissues and in paired adjacent non-neoplastic tissues from patients with ESCC. We evaluated the antitumor effects of lapatinib alone or in combination with oxaliplatin or 5-fluorouracil (5-FU) on a panel of primary ESCC cells in vitro with various levels of EGFR and HER2 expression. The in vivo effect of lapatinib alone or in combination with oxaliplatin or 5-FU was evaluated using a primary ESCC xenograft model. EGFR was overexpressed in 80.9% (76/94) of the ESCC samples, while 24.5% (23/94) of the samples overexpressed HER2. EGFR and HER2 co-overexpression was detected in 22.3% of samples (21/94). In vitro, the primary ESCC cells were more sensitive to lapatinib combined with 5-FU or oxaliplatin than to lapatinib alone. Lapatinib in combination with 5-FU had more potent antitumor effects in the primary ESCC xenograft model, and markedly reduced the phosphorylation of EGFR and HER2, compared with lapatinib alone or in combination with oxaliplatin. These data indicate that lapatinib has activity in EGFR- and/or HER2-expressing ESCC primary cells, and that lapatinib in combination with 5-FU may be a promising treatment strategy for patients with ESCC.

Phosphatase and tensin homolog (PTEN) is a tumor suppressor involved in multiple cell processes. To investigate the role of PTEN in the development of gastric carcinoma, we determined the expression pattern of PTEN in primary gastric carcinoma and in paired adjacent non-neoplastic tissue. We also determined the correlation of PTEN expression with clinicopathological characteristics and patient survival. Overall, 159 gastric carcinomas and 151 paired adjacent non-neoplastic tissues were used in the present study. PTEN expression was determined using tissue microarrays and immunohistochemistry. The clinical sensitivity and specificity of PTEN expression were calculated using receiver operator characteristic curves. Results showed that the loss of cytoplasmic PTEN was significantly more frequent in carcinoma tissue compared with adjacent non-neoplastic tissue (62 vs. 5%, respectively; P

Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G1 state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes. Mol Cancer Ther; 9(7); 2079–89. ©2010 AACR.

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN−/− MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel. [Cancer Res 2009;69(21):8275–83]

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in” PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3β target gene CYCLIN D1 . Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA . Our findings suggest GSK3β is an important effector of mutant PIK3CA , and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.

The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and transgenic mutant ras overexpression prompted us to evaluate the consequences of targeted knock-in of an oncogenic K-ras mutation in the nontumorigenic human breast epithelial cell line MCF-10A and hTERT-immortalized human mammary epithelial cells. Our results show several significant differences between mutant K-ras knock-in cells versus their transgene counterparts, including limited phosphorylation of the downstream molecules extracellular signal-regulated kinase and AKT, minor proliferative capacity in the absence of an exogenous growth factor, and the inability to form colonies in semisolid medium. Analysis of 16 cancer cell lines carrying mutant K-ras genes indicated that 50% of cancer cells harbor nonoverexpressed heterozygous K-ras mutations similar to the expression seen in our knock-in cell lines. Thus, this system serves as a new model for elucidating the oncogenic contribution of mutant K-ras as expressed in a large fraction of human cancer cells. [Cancer Res 2007;67(18):8460–7]

RNA interference (RNAi) has become a popular tool for analyzing gene function in cancer research. The feasibility of using RNAi in cellular and animal models as an alternative to conventional gene knock out approaches has been demonstrated. Although these studies show that RNAi can recapitulate phenotypes seen in knock out animals and their derived cell lines, a systematic study rigorously comparing downstream effector genes between RNAi and gene knock out has not been performed. Here we present data contrasting the phenotypic and genotypic changes that occur with either stable knock down via RNAi of the cyclin dependent kinase inhibitor p21 versus its somatic cell knock out counterpart in the human mammary epithelial cell line MCF-10A. Our results demonstrate that p21 knock down clones display a growth proliferative response upon exposure to Transforming Growth Factor-Beta Type 1 (TGFbeta) similar to p21 knock out clones. However, gene expression profiles were significantly different in p21 knock down cells versus p21 knock out clones. Importantly p21 knock down clones did not display increased gene expression of interleukin-1alpha (IL-1alpha), a critical effector of this growth response previously validated in p21 knock out cells. We conclude that gene knock out can yield additional vital information that may be missed with gene knock down strategies.

The Runt box domain DNA-binding transcription factors (RUNX) play key roles in hematopoietic, bone, and gastric development. These factors regulate angiogenesis and tumorigenic events, functioning as either activators or repressors of target genes. Although RUNX2 is an essential bone maturation factor, it has also been found to promote transformation in vivo and cell proliferation in vitro, perhaps by associating with specific coactivators or corepressors. Adenoviral-mediated overexpression of dominant negative RUNX2 or specific reduction of RUNX2 with RNA-interference inhibits cell proliferation. To determine whether RUNX2 also plays a role in cell transformation, RUNX2 interactions with the coactivator Yes-associated protein (YAP65) were examined. RUNX2 associated with YAP65 via a proline-rich segment in the C-terminal domain (PPPY) and coexpression of RUNX2 and YAP65 significantly increased foci formation and anchorage-independent growth relative to each factor alone. However, in contrast to wild-type RUNX2, a mutant RUNX2(P409A), which does not bind YAP65, did not cooperate with YAP65 to promote anchorage-independent growth. RUNX2 is a strong repressor of the cyclin-dependent kinase inhibitor p21(CIP1), which is known to mediate cell transformation. Overexpression of YAP65 prevented RUNX2-dependent downregulation of p21(CIP1) protein expression while promoting cell transformation. The RUNX2(P409A) mutant retained the ability to bind DNA and repress the p21(CIP1) promoter as shown by DNA precipitation and luciferase-reporter assays, respectively, but was not able to relieve repression of the p21(CIP1) promoter. Therefore, these results reveal a novel function of the RUNX2 and YAP65 interaction in oncogenic transformation that may be mediated by modulation of p21(CIP1) protein expression.

The use of gene therapy to correct mutated or lost gene function for the treatment of human cancers has been an active, yet problematic area of biomedical research. Many technical difficulties, including efficient tissue-specific delivery, integration site specificity and general toxicity, are being addressed. Little is known, however, about the genetic and phenotypic stability that accompanies a successful gene-specific targeting event in a cancer cell. This question was addressed following the creation of a colon cancer cell line in which a mutated hMLH1 gene was corrected via targeted homologous recombination. This correction resulted in the expression of wild-type hMLH1 protein, restoration of the hPMS2 protein and mismatch repair (MMR) proficiency. One of two hMLH1-corrected clones, however, was found to retain defects in MMR activity. These cells continued to express the corrected hMLH1 protein, but had lost expression of another MMR protein, hMSH6. DNA sequence analysis of the hMSH6 gene revealed biallelic expansions of a cytosine repeat region in exon 5 that result in frameshifts leading to premature stop codons. These findings suggest that, similar to acquired drug resistance, the presence of genetically heterogeneous cancer cell populations or acquisition of compensatory mutations can result in 'resistance' to gene replacement therapy.

The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of ∼90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.

Currently, a number of breast cancer cell lines exist that serve as models for both estrogen receptor alpha (ERalpha) positive and ERalpha negative disease. Models are also available for pre-neoplastic breast epithelial cells that do not express ERalpha; however, there are no ideal systems for studying pre-neoplastic cells that are ERalpha positive. This has been largely due to the inability to establish an estrogen growth stimulated, non-tumorigenic breast epithelial cell line, as most human breast epithelial cells engineered to overexpress ERalpha have been found to be growth inhibited by estrogens. We have developed independently derived clones from the non-cancerous MCF-10A human breast cell line that express ERalpha and are growth stimulated by 17-beta-estradiol (E2) in the absence of epidermal growth factor (EGF), a cytokine normally required for MCF-10A cell proliferation. This effect is blocked by the selective estrogen receptor modulator (SERM), Tamoxifen and the selective estrogen receptor downregulator, ICI 182,780 (Faslodex, Fulvestrant). Exposure of these cells to EGF and E2 results in a growth inhibitory phenotype similar to previous reports. These data present a reconciling explanation for the previously described paradoxical effects of ERalpha overexpression, and provide a model for examining the carcinogenic effects of estrogens in non-tumorigenic human breast epithelial cells.

It is now well established that cancer is a genetic disease and that somatic mutations of oncogenes and tumour suppressor genes are the initiators of the carcinogenic process. The phosphatidylinositol 3-kinase signalling pathway has previously been implicated in tumorigenesis, and evidence over the past year suggests a pivotal role for the phosphatidylinositol 3-kinase catalytic subunit, PIK3CA, in human cancers. In this review, we analyse recent reports describing PIK3CA mutations in a variety of human malignancies, and discuss their possible implications for diagnosis and therapy.

A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells. (Cancer Res 2006; 66(2): 682-92)

Transforming growth factor beta type I (TGF-beta) is a ubiquitous cytokine that is well known for its ability to inhibit epithelial cell proliferation. Somatic mutations abrogating the TGF-beta signal transduction pathway are found in many gastrointestinal cancers, confirming its importance as a tumor suppressor. In contrast, many nongastrointestinal epithelial malignancies lack these somatic alterations, yet these cancers still acquire resistance to the growth-inhibitory effects of TGF-beta. In many instances, this resistance is part of a signaling switch whereby TGF-beta loses its growth inhibitory effects and is then used by the epithelial cell in a growth-promoting fashion. The mechanisms that underlie this change in the phenotypic growth response to TGF-beta are now being elucidated. This review focuses on recent advances in understanding the dual nature of the TGF-beta pathway as it relates to human carcinogenesis.Elucidating the molecular basis that enables epithelial cells to change from a growth-suppressive to growth-stimulatory phenotype on TGF-beta exposure is an area of active research. Besides enhancing cancer cell growth, TGF-beta is also thought to promote a malignant cell's ability to metastasize by mediating changes in the cytoskeletal architecture, known as an epithelial-to-mesenchymal transition. This process enables a cancer cell to invade and spread to distal sites. Strong evidence has now emerged suggesting that the ability of a cell to use TGF-beta as a growth-promoting/invasive cytokine is a result of a number of different cellular and nuclear factors, including the absence or disruption of cyclin-dependent kinase inhibitors. This imbalance in cell cycle regulators may be the key element that dictates a cell's response to TGF-beta as growth-inhibitory versus growth-stimulatory, thus explaining the dual nature of TGF-beta signaling.Current studies are beginning to shed light on the mechanisms that allow some nongastrointestinal epithelial cancers to evade the growth inhibitory effects of TGF-beta while simultaneously using this cytokine for growth advantage. By dissecting this phenotypic switch during tumor development, important genes, proteins, and pathways that are involved with TGF-beta signaling continue to be discovered. Knowledge of how premalignant cells and tumor cells respond to the growth promoting effects of TGF-beta and the genes that regulate this process will aid in the development of novel therapeutics and treatment strategies.

Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.

Gene function in cancer can be disrupted either through genetic alterations, which directly mutate or delete genes, or epigenetic alterations, which alter the heritable state of gene expression. The latter events are mediated by formation of transcriptionally repressive chromatin states around gene transcription start sites and an associated gain of methylation in normally unmethylated CpG islands in these regions. The genes affected include over half of the tumor suppressor genes that cause familial cancers when mutated in the germline; the selective advantage for genetic and epigenetic dysfunction in these genes is very similar. The aberrant methylation can begin very early in tumor progression and mediate most of the important pathway abnormalities in cancer including loss of cell cycle control, altered function of transcription factors, altered receptor function, disruption of normal cell-cell and cell-substratum interaction, inactivation of signal transduction pathways, loss of apoptotic signals and genetic instability. The active role of the aberrant methylation in transcriptional silencing of genes is becoming increasingly understood and involves a synergy between the methylation and histone deacetylase (HDAC) activity. This synergy can be mediated directly by HDAC interaction with DNA methylating enzymes and by recruitment through complexes involving methyl-cytosine binding proteins. In the translational arena, the promoter hypermethylation changes hold great promise as DNA tumor markers and their potentially reversible state creates a target for cancer therapeutic strategies involving gene reactivation.

One of the hallmarks of cancer is metabolic deregulation. Many tumors display increased glucose uptake and breakdown through the process of aerobic glycolysis, also known as the Warburg effect. Less studied in cancer development and progression is the importance of the glutamine (Gln) pathway, which provides cells with a variety of essential products to sustain cell proliferation, such as ATP and macromolecules for biosynthesis. To this end Gln dependency was assessed in a panel of non-small cell lung cancer lines (NSCLC). Gln was found to be essential for the growth of cells with high rates of glutaminolysis, and after exploring multiple genes in the Gln pathway, GLS1 was found to be the key enzyme associated with this dependence. This dependence was confirmed by observing the rescue of decreased growth by exogenous addition of downstream metabolites of glutaminolysis. Expression of the GLS1 splice variant KGA was found to be decreased in tumors compared with normal lung tissue. Transient knock down of GLS1 splice variants indicated that loss of GAC had the most detrimental effect on cancer cell growth. In conclusion, NSCLC cell lines depend on Gln for glutaminolysis to a varying degree, in which the GLS1 splice variant GAC plays an essential role and is a potential target for cancer metabolism-directed therapy.

We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I–II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.

Purpose: We sought to evaluate the feasibility of detecting PIK3CA mutations in circulating tumor DNA (ctDNA) from plasma of patients with metastatic breast cancer using a novel technique called BEAMing. Experimental Design: In a retrospective analysis, 49 tumor and temporally matched plasma samples from patients with breast cancer were screened for PIK3CA mutations by BEAMing. We then prospectively screened the ctDNA of 60 patients with metastatic breast cancer for PIK3CA mutations by BEAMing and compared the findings with results obtained by screening corresponding archival tumor tissue DNA using both sequencing and BEAMing. Results: The overall frequency of PIK3CA mutations by BEAMing was similar in both patient cohorts (29% and 28.3%, respectively). In the retrospective cohort, the concordance of PIK3CA mutation status by BEAMing between formalin-fixed, paraffin-embedded (FFPE) samples and ctDNA from temporally matched plasma was 100% (34 of 34). In the prospective cohort, the concordance rate among 51 evaluable cases was 72.5% between BEAMing of ctDNA and sequencing of archival tumor tissue DNA. When the same archival tissue DNA was screened by both sequencing and BEAMing for PIK3CA mutations (n = 41 tissue samples), there was 100% concordance in the obtained results. Conclusions: Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible. Our results suggest that PIK3CA mutational status can change upon disease recurrence, emphasizing the importance of reassessing PIK3CA status on contemporary (not archival) biospecimens. These results have implications for the development of predictive biomarkers of response to targeted therapies. Clin Cancer Res; 18(12); 3462–9. ©2012 AACR.

International audience; Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. Mol Cancer Ther; 11(3); 720–9. ©2011 AACR.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1 's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic “hit” contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Background Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916. Methods 59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response. Results 20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test). Conclusions High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.

The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. To evaluate the biological and clinical relevance of p53 loss, human somatic cell gene targeting was used to delete the TP53 gene in the non-tumorigenic epithelial cell line, MCF-10A. In all four p53−/− clones generated, cells acquired the capability for epidermal growth factor-independent growth and were defective in appropriate downstream signaling and cell cycle checkpoints in response to DNA damage. Interestingly, p53 loss induced chromosomal instability leading to features of transformation and the selection of clones with varying phenotypes. For example, p53-deficient clones were heterogeneous in their capacity for anchorage-independent growth and invasion. In addition, and of clinical importance, the cohort of p53-null clones showed sensitivity to chemotherapeutic interventions that varied depending not only on the type of chemotherapeutic agent, but also on the treatment schedule. In conclusion, deletion of the TP53 gene from MCF-10A cells eliminated p53 functions, as well as produced p53−/− clones with varying phenotypes possibly stemming from the distinct chromosomal changes observed. Such a model system will be useful to further understand the cancer-specific phenotypic changes that accompany p53 loss, as well as help to provide future treatment strategies for human malignancies that harbor aberrant p53.

Preclinical cellular response profiling of tumor models has become a cornerstone in the development of novel cancer therapeutics. As efforts to predict clinical efficacy using cohorts of in vitro tumor models have been successful, expansive panels of tumor-derived cell lines can recapitulate an “all comers” efficacy trial, thereby identifying which tumors are most likely to benefit from treatment. The response profile of a therapy is most often studied in isolation; however, drug treatment effect patterns in tumor models across a diverse panel of compounds can help determine the value of unique molecular target classes in specific tumor cohorts. To this end, a panel of 19 compounds was evaluated against a diverse group of cancer cell lines (n = 311). The primary oncogenic targets were a key determinant of concentration-dependent proliferation response, as a total of five of six, four of four, and five of five phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, insulin-like growth factor-I receptor (IGF-IR), and mitotic inhibitors, respectively, clustered with others of that common target class. In addition, molecular target class was correlated with increased responsiveness in certain histologies. A cohort of PI3K/AKT/mTOR inhibitors was more efficacious in breast cancers compared with other tumor types, whereas IGF-IR inhibitors more selectively inhibited growth in colon cancer lines. Finally, specific phenotypes play an important role in cellular response profiles. For example, luminal breast cancer cells (nine of nine; 100%) segregated from basal cells (six of seven; 86%). The convergence of a common cellular response profile for different molecules targeting the same oncogenic pathway substantiates a rational clinical path for patient populations most likely to benefit from treatment. Cancer Res; 70(9); 3677–86. ©2010 AACR.

Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21's cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-α, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.

Inactivation of the Fanconi anemia (FA) pathway occurs in diverse human tumors among the general population and renders those tumors hypersensitive to DNA interstrand-cross-linking (ICL) agents. The identification of novel agents to which FA pathway–deficient cells were hypersensitive could provide new therapeutic opportunities and improve our molecular understanding of the FA genes. Using high-throughput screening, we assessed the growth of isogenic human cancer cells that differed only in the presence or absence of single FA genes upon treatment with 880 active drugs and 40,000 diverse compounds. We identified several compounds to which FA pathway–deficient cells were more sensitive than FA pathway–proficient cells, including two groups of structurally related compounds. We further investigated the compound eliciting the strongest effect, termed 80136342. Its mechanism of action was distinct from that of ICL agents; 80136342 did not cause increased chromosomal aberrations, enhanced FANCD2 monoubiquitination, H2AX phosphorylation, p53 activation, or ICL induction. Similar to ICL agents, however, 80136342 caused a pronounced G2 arrest in FA pathway–deficient cells. When applied in combination with ICL agents, 80136342 had at least additive toxic effects, excluding interferences on ICL-induced toxicity and facilitating a combinational application. Finally, we identified one particular methyl group necessary for the effects of 80136342 on FA–deficient cells. In conclusion, using high-throughput screening in an isogenic human FA cancer model, we explored a novel approach to identify agents eliciting hypersensitivity in FA pathway–deficient cells. We discovered several attractive candidates to serve as lead compounds for evaluating structure-activity relationships and developing therapeutics selectively targeting FA pathway–deficient tumors. [Cancer Res 2007;67(5):2169–77]

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis. (Cancer Res 2006; 66(18): 8994-9001)

Transforming growth factor-beta type 1 (TGF-beta) has been implicated as both a tumor suppressor and a tumor promoter in many solid epithelial cancers. We have previously demonstrated that the cyclin dependent kinase (CDK) inhibitor p21 acts as a molecular switch in determining a growth inhibitory versus growth proliferative response to TGF-beta in the spontaneously immortalized human mammary epithelial cell line MCF-10A. We now demonstrate that this proliferative effect of TGF-beta is mediated through the proinflammatory cytokine, interleukin-1alpha (IL-1alpha). Using gene expression array analysis, we identified IL-1alpha as a cytokine specifically upregulated only in cells lacking p21 and only upon TGF-beta stimulation. Cell proliferation assays verified that recombinant IL-1alpha was capable of inducing a growth proliferative response in p21 null MCF-10A cells, while neutralizing antibodies against IL-1alpha prevented the growth proliferative effects of TGF-beta. Mechanistically, both the CDK and proliferating cell nuclear antigen (PCNA) inhibitory functions of p21 were responsible for preventing TGF-beta induced cell proliferation, but only PCNA inhibition by p21 regulated IL-1alpha gene expression. These studies demonstrate a novel role for IL-1alpha in mediating a proliferative response to TGF-beta signaling, and suggest that therapies directed against IL-1alpha could abate the growth proliferative effects of TGF-beta without compromising its tumor suppressive function.

The dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is a heterocyclic amine and is a common byproduct of cooked meat and fish. Although most cells undergo apoptosis when exposed to this mutagen, subsets develop resistance. Rather than die, these resistant cells persist and accumulate mutations, thereby driving tumorigenesis of exposed organs within the gastrointestinal tract. By applying a high-throughput cell-based screen of 32,000 small molecules, we have identified a family of compounds that specifically inhibit the growth of PhIP-resistant cancer cells. These compounds may prove useful for the treatment or prevention of gastrointestinal tumors arising after exposure to PhIP and related carcinogens.

3513 Background: In contrast to BRAF-mutant melanoma, the inhibition of BRAF or BRAF/MEK has been relatively ineffective in the treatment of BRAF-mutant CRC, possibly due to EGFR-dependent resistan...

Cancer is a process driven by the accumulation of abnormalities in gene function. While many of these changes are genetic, epigenetically mediated changes in gene expression are being increasingly appreciated. This latter process emphasizes the need to understand two key components of heritable, but reversible, modulation of gene promoter function that are closely tied to one another – formation of chromatin which modulates transcription and establishing patterns of DNA methylation. The link lies first in the recruitment to methylated cytosines of a family of methyl-CpG binding domain proteins (MBDs), which are direct transcriptional repressors and can complex with transcriptional corepressors including histone deacetylases (HDACs). Additionally, the proteins that catalyze DNA methylation, DNA methyltransferases (DNMTs), also directly repress transcription and associate with HDACs. Regulation of these above chromatin-DNA methylation interactions as a function of DNA replication timing is emerging as a key event in the inheritance of transcriptionally repressed domains of the genome. Importantly, synergy between HDAC activity and DNA methylation is operative for a key epigenetic abnormality in cancer cells, transcriptional silencing of tumor suppressor genes. This change has now been recognized for genes that are essential for normal regulation of virtually every major cell function including cell growth, differentiation, apoptosis, DNA repair, and cell–cell, cell–substratum interaction. Understanding the molecular determinants of both normal and abnormal patterns of chromatin formation and DNA methylation thus holds great promise for our understanding of cancer and for means to better diagnose, prevent, and treat this disease.

The phosphoinositide 3-kinase (PI3K) signalling pathway is activated in a broad spectrum of human cancers. Activation of this pathway often occurs indirectly by somatic aberrations of receptor tyrosine kinases, or directly by mutations in PI3K genes, such as PIK3CA. GSK2126458 is a novel, orally administered inhibitor of wild type phosphoinositide 3-kinase alpha (PI3K) and the common activation mutants of p110 found in human cancers that has recently entered Phase I clinical trials. In order to determine histological and genetic profiles of patients that are more likely to gain clinical benefit from GSK2126458, the compound was tested in a 3-day continuous exposure proliferation assay on a panel of 198 genetically characterized human cancer cell lines derived from a diversity of tissue types. Cells originating from bladder, breast, and ovarian cancers are most responsive based on growth inhibition metrics (gIC50's). Breast cancer cell lines harboring activating mutations of PIK3CA are associated with increased levels of response (p = 0.027); an observation which was validated in an independent cohort of 53 breast cell lines (p = 0.021). While colon cancers exhibit an intermediate level of sensitivity compared to other tumor types, specific exon 5 G12/13 hotspot mutations of the KRAS gene are predictive of decreased cellular response (p = 0.025). Additionally, a comparison of these cell line proliferation data to a similarly-assayed panel of 19 compounds of diverse target class shows that while the overall pattern of cellular response to GSK2126458 resembles other inhibitors or the PI3K/AKT/mTOR pathway, distinctions emerge that correlate with pathway targeting strategy. For example, while breast cancers are preferentially responsive to GSK2126458 and other inhibitors of this pathway, cell lines harboring PTEN mutations are less responsive to GSK2126458 than to compounds directly targeting AKT. Collectively these data suggest a unique profile for GSK2126458 among compounds that inhibit the PI3K/AKT/mTOR pathway. Positive predictors of response, such as PIK3CA mutations in breast cancer, may identify patients who are most likely to benefit from GSK2126458. The use of this predictor to select patients has the potential to reduce the size of a clinical registration trial for GSK2126458 by approximately 25%, thereby limiting cost and accelerating the development of this compound. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B140.

Centromere protein-E (CENP-E) is a mitotic kinesin that is required during mitosis for metaphase chromosome alignment. GSK923295 (′295) is a potent and specific inhibitor of human CENP-E currently being developed as a next generation antimitotic drug for the treatment of cancer. It has shown a broad spectrum of anti-cancer activity in vitro and in vivo. A siRNA library screen targeting the ‘druggable genome’ identified multiple MAPK pathway genes as sensitizers to ′295, including BRAF and ERK1. Since the MEK protein plays an important role in the MAPK pathway, combination studies between GSK MEK inhibitor GSK1120212 (′212) and ′295 were performed in cancer cell lines from different tissue origins, and the combination effect was evaluated based on three different criteria: Excess over Single Highest Agent (EOHSA), Bliss, and Combination Index (CI).. Synergistic response was observed in 5/6 colon cancer cell lines, 5/15 lung cancer cell lines, and 6/8 pancreas cancer cell lines. Additionally, the combination of ′212 and ′295 treatment induced a much stronger apoptotic response than either drug alone in two colon cancer cell lines. These data suggest that combining MEK and CENP-E inhibitors might improve clinical efficacy over either as a single agent in selected tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B173.

e14641 Background: Mutations of PIK3CA, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), are found in ∼25% of breast cancers. The tumor suppressor PTEN reverses the effects of PI3K and genetic alterations leading to loss or reduced function of PTEN also occur at high frequency in breast cancers. We have used somatic cell gene targeting to create paired isogenic cell lines with critical genetic alterations as their only differentiating factor. Our lab has introduced two “hotspot” oncogenic PIK3CA mutations into the MCF-10A human breast epithelial cell line. This study led to the discovery that lithium, an FDA approved therapy for bipolar disorders, has selective anti-neoplastic properties against human breast cancer cell lines containing oncogenic PIK3CA mutations. Due to the opposing interactions between PI3K and PTEN, we are now studying whether loss of PTEN also confers sensitivity to lithium using human somatic cell knock outs. Methods: Targeting vectors were created and used to knock out the PTEN gene in MCF-10A. Biochemical and phenotypical characterizations were performed. Paired isogenic cell lines were exposed to lithium to evaluate heterozygous and homozygous loss of PTEN as a predictive biomarker of response. Proteomic and phenotypic changes were measured and assessed. Results: Homozygous loss of PTEN (PTEN-/-) resulted in a transformed phenotype enabling growth in the absence of exogenous growth factors, a hallmark of malignant potential. Preliminary results suggest that treatment of PTEN-/- cells with lithium leads to a decrease in cell proliferation with selectivity for mutant PTEN knock out cell lines, although the effects are not as pronounced as that seen with mutant PIK3CA knock in cells. Conclusions: Our preliminary results suggest that loss of PTEN predicts for sensitivity to lithium in breast cell lines in a manner similar to that seen with oncogenic PIK3CA. We hypothesize that differences in the magnitude of response to lithium may be the result of varied levels of non-PI3K-AKT-mTOR activation between the PIK3CA knock in cell lines and the PTEN knock out cells. No significant financial relationships to disclose.

We attempted to answer two central questions about epigenetic silencing of the tumor suppressor gene p16INK4a in this study: (1) whether the maintenance of associated histone modifications is dependent on DNA methylation and (2) whether such histone modifications can occur prior to DNA methylation. By coupling chromatin immunoprecipitation with gene targeting and the analysis of specific alleles, we found that elimination of DNA methylation from a p16INK4a allele resulted in profound changes in surrounding histones. After continued passage of such cells, methylation of histone H3 lysine-9 occurred in conjunction with re-silencing in the absence of DNA methylation. These results have important implications for understanding the biochemical events underlying the silencing of tumor suppressor genes and the resultant growth suppression.