Tumorigenesis In Mouse Model Research Articles

Prediction and Testing of Biological Networks Underlying Intestinal Cancer

Colorectal cancer progresses through an accumulation of somatic mutations, some of which reside in so-called “driver” genes that provide a growth advantage to the tumor. To identify points of intersection between driver gene pathways, we implemented a network analysis framework using protein interactions to predict likely connections – both precedented and novel – between key driver genes in cancer. We applied the framework to find significant connections between two genes, Apc and Cdkn1a (p21), known to be synergistic in tumorigenesis in mouse models. We then assessed the functional coherence of the resulting Apc-Cdkn1a network by engineering in vivo single node perturbations of the network: mouse models mutated individually at Apc (Apc1638N+/−) or Cdkn1a (Cdkn1a−/−), followed by measurements of protein and gene expression changes in intestinal epithelial tissue. We hypothesized that if the predicted network is biologically coherent (functional), then the predicted nodes should associate more specifically with dysregulated genes and proteins than stochastically selected genes and proteins. The predicted Apc-Cdkn1a network was significantly perturbed at the mRNA-level by both single gene knockouts, and the predictions were also strongly supported based on physical proximity and mRNA coexpression of proteomic targets. These results support the functional coherence of the proposed Apc-Cdkn1a network and also demonstrate how network-based predictions can be statistically tested using high-throughput biological data.

Abstract 3789: Elevated inorganic phosphate stimulates immediate early gene expression and requires FGF receptor signaling

Abstract Dietary inorganic phosphate represents a novel chemoprevention target. Recent results from two different mouse models of tumorigenesis; the two-stage skin carcinogenesis and KrasLA1 lung tumor models suggest that a high phosphate diet increases tumor/papilloma number by approximately 50% relative to a low phosphate diet. Additionally, a low phosphate diet resulted in delayed incidence and growth of papillomas. These results suggest that increased consumption of dietary inorganic phosphate may have long-term consequences on overall health and particularly to tumorigenesis and consequently reducing dietary phosphate intake may be a novel chemoprevention strategy. Our in vitro studies have revealed an active role of inorganic phosphate in altering cell function in various cell types and suggest that the amount of available inorganic phosphate may have significant cell autonomous consequences on cell behavior including activation of ras and ERK1/2 signaling, and increased expression of transformation and metastasis associated genes such as osteopontin (spp1). However, the mechanism by which cells sense and respond to changes in extracellular phosphate resulting in increased proliferation and transformation is not fully understood. We have performed an analysis of the temporal changes in gene expression in response to elevated phosphate. Results identified the strong stimulation of immediate early genes within 15 minutes of exposure to elevated phosphate. Reporter and DNA binding assays revealed the requirement of serum response and AP-1 transcriptional elements and taken together characterize phosphate as a novel mitogen. Furthermore, we have identified the requirement of FGF receptor signaling as one of the earliest events in the phosphate signaling cascade. This finding provides a novel link between growth factor signaling, nutrient sensing and cell growth and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3789.

Abstract LB-42: PDK1 knockdown selectively inhibits growth of cancer cells harboring activating mutations involved in MAPK signaling

Abstract Phosphoinositide-dependent protein kinase-1 (PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway which is the most commonly deregulated signaling pathway across all cancers. Increased PDK1 activity confers chemoresistance in tumor cell lines, and reduced PDK1 expression prevents the onset of tumorigenesis in mouse models. To demonstrate that PDK1 is a promising target for cancer therapy, we used a siRNA-based approach to determine the effects of PDK1 knockdown in cancer cells and primary early-passage normal cells. Loss of PDK1 protein caused a significant reduction in phosphorylation of two PDK1 substrates, AKT (T308) and RSK1 (S221). Interestingly, knockdown of PDK1 protein had preferential growth inhibitory effects in cancer cells containing activating mutations of KRAS or BRAF, compared to cells with wild-type KRAS or BRAF. In KRAS/BRAF-mutant cells, apoptosis was evidenced by PARP-1 cleavage and an increase in the number of sub-G1 DNA content. Overexpression of mouse PDK1 rescued these cells from apoptosis, confirming the specificity of the siRNA-mediated effects. Consistent with these observations, stable expression of mutant KRAS (G12V) in NL-20 immortalized normal lung epithelial cells sensitized these cells to apoptosis upon knockdown of the PDK1 protein. Importantly, overexpression of a kinase-dead mouse PDK1 mutant did not rescue cells from apoptosis, suggesting that the kinase activity is required for these PDK1 functions. Based on these results, we sought to identify inhibitors of the kinase activity of PDK1 for development as anticancer agents. GSK2334470 was identified as a potent inhibitor of PDK1 kinase (IC50 0.5 nmol/l) that demonstrated a high level of specificity to PDK1 in an in vitro kinase panel. In cell-based mechanistic assays, GSK2334470 effectively inhibited phosphorylation of the PDK1-dependent phosphorylation sites [AKT T308 (IC50: 100 nmol/L) and RSK S221 (IC50: 291 nmol/L)], but not the PDK1-independent phosphorylation site on AKT, S473 (IC50: >30 umol/L). Antiproliferative effects were seen in several cell lines using low micromolar concentrations of inhibitor; however, no correlation between growth inhibition and KRAS/BRAF mutations was observed. Further optimization may be necessary for improved potency and KRAS/BRAF selectivity. Currently, efforts are underway to better understand the disconnect between siRNA data and compound data in several experimental cancer models, particularly in those where KRAS or BRAF mutations predominate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-42.

Exploring the Gain of Function Contribution of AKT to Mammary Tumorigenesis in Mouse Models

Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers, mainly as a consequence of the PTEN/PI3K pathway deregulation. To investigate the mechanistic basis of the AKT gain of function-dependent mechanisms of breast tumorigenesis, we explored the phenotype induced by activated AKT transgenes in a quantitative manner. We generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland. We thoroughly analyzed the preneoplastic and neoplastic mammary lesions of these mice and correlated the process of tumorigenesis to AKT levels. Finally, we analyzed the impact that a possible senescent checkpoint might have in the tumor promotion inhibition observed, crossing these lines to mammary specific p53(R172H) mutant expression, and to p27 knock-out mice. We analyzed the benign, premalignant and malignant lesions extensively by pathology and at molecular level analysing the expression of proteins involved in the PI3K/AKT pathway and in cellular senescence. Our findings revealed an increased preneoplastic phenotype depending upon AKT signaling which was not altered by p27 or p53 loss. However, p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinoma observed, but was not sufficient to promote full penetrance of the tumorigenic phenotype. Molecular analysis suggest that tumors from double myrAKT;p53(R172H) mice result from acceleration of initiated p53(R172H) tumors and not from bypass of AKT-induced oncogenic senescence. Our work suggests that tumors are not the consequence of the bypass of senescence in MIN. We also show that AKT-induced oncogenic senescence is dependent of pRb but not of p53. Finally, our work also suggests that the cooperation observed between mutant p53 and activated AKT is due to AKT-induced acceleration of mutant p53-induced tumors. Finally, our work shows that levels of activated AKT are not essential in the induction of benign or premalignant tumors, or in the cooperation of AKT with other tumorigenic signal such as mutant p53, once AKT pathway is activated, the relative level of activity seems not to determine the phenotype.

Abstract B272: The novel PIM kinase inhibitor SGI-1776 significantly enhances the preclinical activity of sunitinib in renal cell carcinoma

Abstract The PIM kinases (PIM1, PIM2, and PIM3) are constitutively active serine/threonine kinases that promote tumorigenesis in mouse models and are associated with drug resistance. Upregulation of PIM kinase expression has been reported in many malignancies including renal cell carcinoma (RCC), suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. SGI-1776 is a novel orally available small molecule inhibitor of PIM kinase activity that is currently in Phase I clinical trials. We hypothesized that inhibition of PIM kinase activity would reduce the viability of RCC cells and enhance the activity of the multi-targeted receptor tyrosine kinase (RTK) inhibitor sunitinib. Cell viability and apoptosis assays demonstrated that SGI-1776 possessed significant in vitro activity in a panel of RCC cell lines. One of the mechanisms by which the PIM kinases have been reported to inhibit apoptosis is by phosphorylating the BH3-only protein Bad to abrogate its pro-apoptotic function. Accordingly, SGI-1776-mediated apoptosis was associated with decreased levels of the phosphorylated form of Bad. Moreover, targeted knockdown of Bad expression significantly decreased sensitivity to SGI-1776 indicating that Bad is an important mediator of the anticancer activity of this agent. Treatment with SGI-1776 also led to a markedly decrease in phosphorylated and total c-Myc levels and enhanced the activity of sunitinib in RCC cells. The addition of sunitinib to SGI-1776 further reduced c-Myc expression. Silencing of the gene suggested that c-Myc plays a significant role in sensitivity to the SGI-1776/sunitinib combination. To further evaluate this drug combination, Caki-1 and 786-O RCC xenografts were established in nude mice and they were treated orally with SGI-1776, sunitinib, or the combination. The SGI-1776/sunitinib combination significantly reduced tumor burden in both xenograft models compared to single agent therapy and was very well tolerated. Analysis of the tumors revealed that the drug combination strongly decreased tumor proliferation and induced apoptosis as measured by PCNA and TUNEL immunohistochemistry, respectively. These data indicate that PIM kinase signaling has a significant role in the biology of RCC and warrants further investigation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B272.

Genetic heterogeneity of Myc-induced mammary tumors reflecting diverse phenotypes including metastatic potential

Human cancers result from a complex series of genetic alterations, resulting in heterogeneous disease states. Dissecting this heterogeneity is critical for understanding underlying mechanisms and providing opportunities for therapeutics matching the complexity. Mouse models of cancer have generally been used to reduce this complexity and focus on the role of single genes. Nevertheless, our analysis of tumors arising in the MMTV-Myc model of mammary carcinogenesis reveals substantial heterogeneity, seen in both histological and expression phenotypes. One contribution to this heterogeneity is the substantial frequency of activating Ras mutations. Additionally, we show that these Myc-induced mammary tumors exhibit even greater heterogeneity, revealed by distinct histological subtypes as well as distinct patterns of gene expression, than many other mouse models of tumorigenesis. Two of the major histological subtypes are characterized by differential patterns of cellular signaling pathways, including beta-catenin and Stat3 activities. We also demonstrate that one of the MMTV-Myc mammary tumor subgroups exhibits metastatic capacity and that the signature derived from the subgroup can predict metastatic potential of human breast cancer. Together, these data reveal that a combination of histological and genomic analyses can uncover substantial heterogeneity in mammary tumor formation and therefore highlight aspects of tumor phenotype not evident in the population as a whole.

Chemoprevention of Mouse Intestinal Tumorigenesis by the Cyclin-Dependent Kinase Inhibitor SNS-032

Despite advances in screening and treatment, colorectal cancer remains the second leading cause of cancer-related death in the United States. Cyclin-dependent kinases (Cdk) are deregulated in colorectal cancer by silencing of the Cdk inhibitor p16(Ink4a) and other mechanisms. We tested whether the small molecule Cdk inhibitor SNS-032 (formerly BMS-387032), which targets Cdk2, Cdk7, and Cdk9, can prevent intestinal tumorigenesis in mouse models. We generated mice with high intestinal tumor loads by combining the multiple intestinal neoplasia (Min) mutation with Ink4a/Arf mutations and inducing colitis with dextran sulfate sodium. p16-null Min mice (n = 17) began dextran sulfate sodium treatment at week 5 and i.p. injection of carrier or SNS-032 at week 6. Mice were sacrificed at week 12. SNS-032 was well tolerated and reduced colon tumor burden to 36% of that in carrier-treated mice (P < 0.001). We then extended the study to Ink4/Arf-null Min mice (n = 14) and increased the drug dose frequency. SNS-032 treatment reduced the intestinal tumor number to 25% and intestinal tumor burden to 16% of carrier-treated mice (P < 0.0001). DNA synthesis in non-neoplastic and tumor epithelial cells, detected by bromodeoxyuridine incorporation, was modestly reduced by acute SNS-032 treatment. The mitotic index, detected by histone H3 phosphorylation, was distinctly decreased (P < 0.03), and apoptosis, detected by caspase 3 activation, was increased (P < 0.005). These results show the chemoprevention of intestinal tumorigenesis by SNS-032. Our findings support further study of Cdk inhibitors for chemoprevention and therapy of colon cancer.

ADAM9 as a Potential Target Molecule in Cancer

ADAM (a disintegrin and metalloproteinase) proteins have a predominant role in the protein ectodomain shedding of membrane-bound molecules. ADAMs have emerged as critical regulators of cell-cell signaling during development and homeostasis, and are believed to contribute to pathologies, such as cancer, where their regulation is altered. ADAM9 is consistently overexpressed in various human cancers, and plays a role in tumorigenesis in mouse models. ADAM9 cleaves and releases a number of molecules with important roles in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin, and could represent a potential therapeutic target in tumors where it is highly expressed. This review provides an overview of ADAM9 with a major focus on its contribution to tumorigenesis. Its role in the shedding of cell surface molecules will be discussed along with emerging aspects of regulation and possible functions in cancer development.

The APC tumor suppressor is required for epithelial integrity in the mouse mammary gland

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor has been associated with mammary tumorigenesis in mouse models and through epidemiological studies of human breast cancers, but the normal role for APC in mammary development has not been thoroughly characterized. We report here that Apc(Min/+) mice containing one functional allele of Apc have severely disrupted lobuloalveolar development during pregnancy and lactation, time points at which Apc gene expression is at its highest levels in normal mice. This phenotype was accompanied by altered proliferation during pregnancy and involution, increased apoptosis throughout lactation, the formation of preneoplastic lesions and changes in specific genes associated with each of these processes. Neither modifications in beta-catenin localization, nor the expression of beta-catenin transcriptional target genes, were observed in Apc(Min/+) mammary tissues; however, tissues from lactating Apc(Min/+) mice had a significantly altered epithelial architecture, including disrupted localization of junctional proteins and polarization. Consistent with these findings, APC knockdown in non-transformed mouse mammary epithelial cells in vitro resulted in altered monolayer formation and proliferation without changes in beta-catenin-mediated transcription. These results suggest that APC expression is tightly regulated during mammary gland development and is required for normal mammary homeostasis and tumor suppression primarily through maintaining epithelial integrity.

The roles of PTEN in development, physiology and tumorigenesis in mouse models: a tissue-by-tissue survey

In 1997, PTEN (phosphatase and tensin homologue deleted on chromosome 10, 10q23.3) was identified as an important tumor suppressor gene that is inactivated in a wide variety of human cancers. Ever since, PTEN's function has been extensively studied, and huge progress has been made in understanding PTEN's role in normal physiology and disease. In this review, we will systematically summarize the important data that have been gained from gene inactivation studies in mice and will put these data into physiological context using a tissue-by-tissue approach. We will cover mice exhibiting complete and constitutive inactivation of Pten as well as a large number of strains in which Pten has been conditionally deleted in specific tissues. We hope to highlight not only the tumor suppressive function of Pten but also its roles in embryogenesis and in the maintenance of the normal physiological functions of many organ systems.

Effects of mutant human Ki- rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki- ras G12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki- ras G12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki- ras G12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.

Reproducibility of Tumor Volume Measurement at MicroCT Colonography in Living Mice

We sought to demonstrate the viability of microcomputed tomographic colonography (muCTC) as a tool for monitoring tumorigenesis in mouse models of human colorectal cancer during prospective longitudinal studies. The precision and accuracy of volumetric measurements were determined to assess whether changes in tumor volume over time were readily detectable. All animal studies were conducted under the guidelines set forth by the Institutional Animal Care and Use Committee of the American Association for Assessment and Accreditation of Laboratory Animal Care. muCTC was performed on C57BL/6J (B6) mice carrying the Min allele of Apc, ultimately yielding 18 scans. Assessments of scan quality and tumor volume were both performed once per week over 8 weeks. Scans with a good quality rating had a mean standard deviation in tumor volume measurement of 8%. By contrast, scans with a poor quality rating had a mean standard deviation in tumor volume measurement of 35%. Variables affecting muCTC scan quality in living mice included bowel preparation, motion artifact, and tumor morphology. Tumor volume measurements were highly correlated with tumor weight (r2 = 0.87). The reproducibility of tumor volume measurement at muCTC in living mice makes prospective longitudinal evaluation of colonic tumor response feasible. For muCTC scans of good quality, a 16% change in tumor volume can be detected at the 95% confidence level.

Fat10 is an epigenetic marker for liver preneoplasia in a drug-primed mouse model of tumorigenesis

There is clinical evidence that chronic liver diseases in which MDBs (Mallory Denk Bodies) form progress to hepatocellular carcinoma. The present study provides evidence that links MDB formation induced by chronic drug injury, with preneoplasia and later to the formation of tumors, which develop long after drug withdrawal. Evidence indicated that this link was due to an epigenetic cellular memory induced by chronic drug ingestion. Microarray analysis showed that the expressions of many markers of preneoplasia (UBD, Alpha Fetoprotein, KLF6 and glutathione- S-transferase mu2) were increased together when the drug DDC was refed. These changes were suppressed by S-adenosylmethionine feeding, indicating that the drug was affecting DNA and histones methylation in an epigenetic manner. The link between MDB formation and neoplasia formation was likely due to the over expression of UBD (also called FAT10), which is up regulated in 90% of human hepatocellular carcinomas. Immunohistochemical staining of drug-primed mouse livers showed that FAT10 positive liver cells persisted up to 4 months after drug withdrawal and they were still found in the livers of mice, 14 months after drug withdrawal. The refeeding of DDC increased the percent of FAT10 hepatocytes.

Wnt signaling and gastrointestinal tumorigenesis in mouse models

The canonical Wnt signaling plays important roles in embryonic development and tumorigenesis. For the latter, induced mutations in mice have greatly contributed to our understanding of the molecular mechanisms of cancer initiation and progression. Here, I will review recent reports on gastrointestinal cancer model mice, with an emphasis on the roles of the Wnt signal pathway. They include: mouse models for familial adenomatous polyposis; modifying factors that affect mouse intestinal polyposis, including the genes that help cancer progression; Wnt target genes that affect mouse intestinal polyposis; and a mouse model of gastric cancer that mimics Helicobacter pyroli infection.

Effect of an Epidermal Growth Factor Receptor Inhibitor in Mouse Models of Lung Cancer

Gefitinib (Iressa, ZD1839) is a potent high-affinity competitive tyrosine kinase inhibitor aimed primarily at epidermal growth factor receptor (EGFR). Inhibitors in this class have recently been approved for clinical use in the treatment of advanced non-small cell lung cancer as monotherapy following failure of chemotherapy. We examined the efficacy of gefitinib on lung tumorigenesis in mouse models using both postinitiation and progression protocols. Gefitinib was given at a dose of 200 mg/kg body weight (i.g.) beginning either 2 or 12 weeks following carcinogen initiation. In the postinitiation protocol, gefitinib significantly inhibited both tumor multiplicity (approximately 70%) and tumor load (approximately 90%) in A/J or p53-mutant mice (P < 0.0001). Interestingly, gefitinib was also highly effective against lung carcinogenesis in the progression protocol when individual animals already have multiple preinvasive lesions in the lung. Gefitinib exhibited approximately 60% inhibition of tumor multiplicity and approximately 80% inhibition of tumor load when compared with control mice (both P < 0.0001). These data show that gefitinib is a potent chemopreventive agent in both wild-type and p53-mutant mice and that a delayed administration was still highly effective. Analyses of mutations in the EGFR and K-ras genes in lung tumors from either control or treatment groups showed no mutations in EGFR and consistent mutation in K-ras. Using an oligonucleotide array on control and gefitinib-treated lesions showed that gefitinib treatment failed to alter the activity or the expression level of EGFR. In contrast, gefitinib treatment significantly altered the expression of a series of genes involved in cell cycle, cell proliferation, cell transformation, angiogenesis, DNA synthesis, cell migration, immune responses, and apoptosis. Thus, gefitinib showed highly promising chemopreventive and chemotherapeutic activity in this mouse model of lung carcinogenesis.

Connecting Senescence to DNA Damage Response

The body has various mechanisms to prevent the progression from preneoplastic lesion to full neoplasia. One of these is oncogene-induced senescence or withdrawal from the cell cycle. Two groups (Bartkova et al . and Di Micco et al .) now provide evidence that certain oncogenes trigger DNA replication stress and activation of the DNA damage checkpoint response and that activation of the checkpoint response is essential for the observed senescence. Bartkova et al . investigated senescence induced by Mos (an activator of the mitogen-activated protein kinase pathway), Cdc6 (a DNA replication licensing factor), and cyclin E (a protein involved in the cell cycle) in various human diploid cells. In each case, the cells exhibited activation of the DNA damage checkpoint and senescence. If the activity of ATM--a kinase critical to the DNA damage checkpoint response--was blocked by siRNA or pharmacological agents, then senescence was prevented. DNA labeling experiments indicated that the oncogenes triggered the formation of prematurely terminated DNA replication forks, which was the likely mechanism by which double-stranded breaks formed that triggered the DNA damage response. Di Micco et al . provide evidence linking the DNA damage checkpoint response to Ras-induced senescence. Cells undergoing oncogene-induced senescence exhibited activation of the DNA damage checkpoint response. Cells in which checkpoint kinase 2 (CHK2) activity was diminished (by knockout or RNA interference) were more susceptible to Ras-induced transformation and continued to proliferate. Ras triggered a proliferative burst and the cells that subsequently underwent senescence exhibited partly replicated DNA. Bartkova et al . found that there is a correlation in colon adenoma samples between the appearance of markers for senescence and activation of the DNA damage checkpoint, and that progression to carcinoma correlated with a decrease in these two processes. Both groups looked at mouse models of tumorigenesis. Bartkova et al . found that injection of a ras-transformed tumor line into mice in which the activity of ATM was decreased led to the formation of larger and more invasive tumors. Di Micco et al . showed that chemicals that are associated with the induction of Ras mutations in mouse skin cause cellular proliferation and activation of the DNA damage response. It appears that the proliferation triggered by oncogenes can lead to DNA stress, which triggers the DNA damage response, allowing the body to prevent tumor progression. J. Bartkova, N. Rezaei, M. Liontos, P. Karakaidos, D. Kletsas, N. Issaeva, L.-V. F. Vassiliou, E. Kolettas, K. Niforou, V. C. Zoumpourlis, M. Takaoka, H. Nakagawa, F. Tort, K. Fugger, F. Johansson, M. Sehested, C. L. Andersen, L. Dyrskjot, T. Ørntoft, J. Lukas, C. Kittas, T. Helleday, T. D. Halazonetis, J. Bartek, V. G. Gorgoulis, Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444 , 633-637 (2006). [PubMed] R. Di Micco, M. Fumagalli, A. Cicalese, S. Piccinin, P. Gasparini, C. Luise, C. Schurra, M. Garré, P. G. Nuciforo, A. Bensimon, R. Maestro, P. G. Pelicci, F. d’Adda di Fagagna, Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444 , 638-642 (2006). [PubMed]

RACK1-mediated Integration of Adhesion and Insulin-like Growth Factor I (IGF-I) Signaling and Cell Migration Are Defective in Cells Expressing an IGF-I Receptor Mutated at Tyrosines 1250 and 1251

The scaffolding protein receptor for activated C kinase (RACK1) has been proposed to mediate the integration of insulin-like growth factor I receptor (IGF-IR) and adhesion signaling. Here we investigated the mechanism of this integration of signaling, by using an IGF-IR mutant (Y1250F/Y1251F) that is deficient in anti-apoptotic and transforming function. RACK1 was found to associate with the IGF-IR only in adherent cells and did not associate with the IGF-IR in nonadherent cells, lymphocytic cells, or cells expressing the Y1250F/Y1251F mutant. In R- cells transiently expressing the Y1250F/Y1251F mutant RACK1 became constitutively associated with beta1 integrin and did not associate with Shc, Src, or Shp2. This was accompanied by the loss of formation of a complex containing the IGF-IR, RACK1, and beta1 integrin; loss of migratory capacity; enhanced Src and FAK activity; enhanced Akt phosphorylation; and decreased p38 mitogen-activated protein kinase activity. Shc was not phosphorylated in response to IGF-I in cells expressing the Y1250F/Y1251F mutant and remained associated with protein phosphatase 2A. Similar alterations in signaling were observed in cells that were stimulated with IGF-I in nonadherent cultures. Our data suggest that disruption of RACK1 scaffolding function in cells expressing the Y1250F/Y1251F mutant results in the loss of adhesion signals that are necessary to regulate Akt activity and to promote turnover of focal adhesions and cell migration.

Global expression profiling identifies signatures of tumor virulence in MMTV-PyMT-transgenic mice: correlation to human disease.

FVB/N-Tg (MMTV-PyMT)(634Mul)-transgenic mice develop multifocal mammary tumors with a high incidence of pulmonary metastasis. We have demonstrated previously that mammary tumors derived from transgene-positive F1 progeny in particular inbred strains display altered latency, tumor growth rates, and metastatic rates when compared with the FVB/NJ homozygous parent. To identify genes with expression that might be critical in modifying the biological behavior of MMTV-PyMT tumors, we performed a detailed comparative analysis of expression profiles from mammary tumors arising in the parental FVB/NJ background and F1 progeny from crosses with I/LnJ, LP/J, MOLF/Ei, and NZB/B1NJ mice. Compared with normal mammary glands, gene expression profiles of tumors from all five strains exhibited up-regulation of genes involved in cell growth (e.g., Cks1 and CDC25C) and down-regulation of cell adhesion molecules, with many genes associated previously with human breast cancer such as STAT2, CD24 antigen, gelsolin, and lipocalin2. To identify genes with significant variation in expression between the five different genotypes, significance analysis of microarrays (SAM) and one-way ANOVA were used. Three definable groupings of tumors were identified: (a) tumors derived in the LP/J F1 and MOLF/Ei F1 strains in which tumor growth and dissemination are suppressed and latency prolonged; (b) the most aggressive tumors from the FVB/NJ parental strain and I/LnJ F1 genomic backgrounds; and (c) an intermediate virulence phenotype with tumors from NZB/B1NJ-F1 crosses. These array based assessments correlated well with a composite phenotype ranking using a "virulence" index. The gene expression signature that is associated with a high metastatic rate in the mouse contains the same 17 genes described recently as the signature gene set predictive of metastasis in human tumors (1) with 16 of the 17 genes exhibiting the same directional change in expression associated with human metastases. These results demonstrate that the genetic analysis of mouse models of tumorigenesis may be highly relevant to human cancer and that the metastatic phenotype of a tumor may be affected by the germline genetic configuration of the host.

A Mouse Model of Uterine Leiomyosarcoma

We are using an approach that is based on the cre/loxP recombination process and involves a binary system of Cre-producing and Cre-responding transgenic mice to achieve ubiquitous or tissue-specific expression of oncoproteins. To develop mouse models of tumorigenesis, Cre-producers are mated with responder animals carrying a dormant oncogene targeted into the 3' untranslated region of the locus encoding cytoplasmic beta-actin (actin cassette). Production of oncoprotein from a bicistronic message is accomplished in bitransgenic progeny by Cre-mediated excision of a segment flanked by loxP sites that is located upstream from the oncogenic sequence. Widespread Cre-dependent activation and expression of an actin-cassette transgene encoding the T antigens of the SV40 early region (SVER) commencing in embryos was compatible with normal development and did not impair viability. However, at approximately 3 months of age, all female animals developed massive uterine leiomyosarcomas, whereas practically all males exhibited enormously enlarged seminal vesicles because of pronounced hyperplasia of the smooth muscle layers. In addition, because of smooth muscle hyperproliferation, marked dilation of the gallbladder was observed in mice of both sexes. To begin exploring aberrant signaling events in the SVER-triggered tumorigenic pathways, we analyzed the expression profile of leiomyosarcomas by DNA microarray analysis.

Restriction landmark genome scanning

Restriction landmark genome scanning (RLGS) is a quantitative approach that is uniquely suited for simultaneously assessing the methylation status of thousands of CpG islands. RLGS separates radiolabeled NotI fragments (or other CpG-containing restriction enzyme fragments) in two dimensions and allows distinction of single-copy CpG islands from multicopy CpG-rich sequences. The methylation sensitivity of the endonuclease activity of NotI provides the basis for differential methylation analysis, and NotI sites occur primarily in CpG islands and genes. RLGS has been used to identify novel imprinted genes, novel targets of DNA amplification and methylation in human cancer, and to identify deletion, methylation, and gene amplification in a mouse model of tumorigenesis. Such massively parallel analyses are critical for pattern recognition within and between tumor types, and for estimating the overall influence of CpG island methylation on the cancer cell genome. RLGS is also a useful method for integrating methylation analyses with high-resolution gene copy number analyses.