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PDF file - 208K, DHT regulated gene pathways in cyclin D1+/+ and cyclin D1-/- mouse prostates.

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PDF file - 111K, Cyclin D1 dependent DHT regulated gene expression.

Initially isolated as the dominant suppressor of the mutant epidermal growth factor receptor (ellipse), the Dachshund gene plays a key role in metazoan development regulating the Retinal Determination Gene Network. Herein, the DACH1 gene was expressed in normal prostate epithelial cells with reduced expression in human prostate cancer. DACH1 inhibited prostate cancer cellular DNA synthesis, growth in colony forming assays, and blocked contact-independent growth in soft agar assays. DACH1 inhibited androgen receptor (AR) activity, requiring a conserved DS Domain (Dachshund domain conserved with Ski/Sno) that bound NCoR/HDAC and was recruited to an androgen-responsive gene promoter. DACH1 inhibited ligand-dependent activity of AR mutations identified in patients with androgen-insensitive prostate cancer. The DS domain was sufficient for repression of the AR wild-type but failed to repress an AR acetylation site point mutant. These studies show a role for the Retinal Determination Gene Network in regulating cellular growth and signaling in prostate cancer. [Cancer Res 2009;69(8):3347–55]

Supplementary Methods from The Canonical NF-κB Pathway Governs Mammary Tumorigenesis in Transgenic Mice and Tumor Stem Cell Expansion

PDF file - 131K, Endogenous cyclin D1 maintains prostate cancer stem cell population.

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Supplementary Figure 1 from The Cell Fate Determination Factor Dachshund Inhibits Androgen Receptor Signaling and Prostate Cancer Cellular Growth

Supplementary Figure Legends 1-5 from The Canonical NF-κB Pathway Governs Mammary Tumorigenesis in Transgenic Mice and Tumor Stem Cell Expansion

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Supplementary Figures 1-5 from The Canonical NF-κB Pathway Governs Mammary Tumorigenesis in Transgenic Mice and Tumor Stem Cell Expansion

XLSX file - 118K, Functional annotation clustering of cyclin D1 induced genes.

XLSX file - 398K, Lists of genes differentially regulated by DHT in cyclin D1+/+ mice, DHT regulated genes in cyclin D1-/- mice and genes differentially regulated by cyclin D1.

Supplementary Figure 2 from The Cell Fate Determination Factor Dachshund Inhibits Androgen Receptor Signaling and Prostate Cancer Cellular Growth

PDF file - 83K, Cyclin D1 is required for prostate development in vivo.

PDF file - 107K, Cyclin D1 expression signature is induced in prostatospheres and induction of Wnt.

Improved clinical management of prostate cancer has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human prostate cancer. The TGF-β/BMP-SMAD4 signaling pathway, which induces epithelial–mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene–transformed prostate cancer cell lines. The in vivo cyclin D1–mediated molecular signature predicted poor outcome of recurrence-free survival for patients with prostate cancer (K-means HR, 3.75, P = 0.02) and demonstrated that endogenous cyclin D1 restrains TGF-β, Snail, Twist, and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In chromatin immunoprecipitation sequencing, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical-free recurrence in human prostate cancer. Cancer Res; 74(2); 508–19. ©2013 AACR.

Supplementary Methods from The Cell Fate Determination Factor Dachshund Inhibits Androgen Receptor Signaling and Prostate Cancer Cellular Growth

Summary DACH1 abundance is reduced in human malignancies, including breast cancer. Herein DACH1 was detected among multipotent fetal mammary stem cells in the embryo, among mixed lineage precursors, and in adult basal cells and (ERα+) luminal progenitors. Dach1 gene deletion at 6 weeks in transgenic mice reduced ductal branching, reduced the proportion of mammary basal cells (Lin− CD24med CD29high) and reduced abundance of basal cytokeratin 5, whereas DACH1 overexpression induced ductal branching, increased Gata3 and Notch1, and expanded mammosphere formation in LA-7 breast cells. Mammary gland-transforming growth factor β (TGF-β) activity, known to reduce ductal branching and to reduce the basal cell population, increased upon Dach1 deletion, associated with increased SMAD phosphorylation. Association of the scaffold protein Smad anchor for receptor activation with Smad2/3, which facilitates TGF-β activation, was reduced by endogenous DACH1. DACH1 increases basal cells, enhances ductal formation and restrains TGF-β activity in vivo., Highlights • Dach1 is expressed in mammary gland fetal stem cells and adult luminal cells • Dach1 expands mammary gland basal/myoepithelial cells • Dach1 induces post-natal mammary gland ductal formation • Dach1 retrains TGF-β activity in the mammary gland in vivo, DACH1 abundance, is reduced in human malignancies, including breast cancer. In this article, Dr. Pestell and his colleagues showed that DACH1 is expressed in multipotent mammary gland fetal stem cells and both the adult basal and ERα+ luminal cells, promotes mammary gland stem cell and basal/myoepithelial cell expansion, promotes mammary gland ductal branching, and restrains TGF-β action in the pubertal mammary gland.

Lysine methylation of histones and non-histone substrates by the SET domain containing protein lysine methyltransferase (KMT) G9a/EHMT2 governs transcription contributing to apoptosis, aberrant cell growth, and pluripotency. The positioning of chromosomes within the nuclear three-dimensional space involves interactions between nuclear lamina (NL) and the lamina-associated domains (LAD). Contact of individual LADs with the NL are dependent upon H3K9me2 introduced by G9a. The mechanisms governing the recruitment of G9a to distinct subcellular sites, into chromatin or to LAD, is not known. The cyclin D1 gene product encodes the regulatory subunit of the holoenzyme that phosphorylates pRB and NRF1 thereby governing cell-cycle progression and mitochondrial metabolism. Herein, we show that cyclin D1 enhanced H3K9 dimethylation though direct association with G9a. Endogenous cyclin D1 was required for the recruitment of G9a to target genes in chromatin, for G9a-induced H3K9me2 of histones, and for NL-LAD interaction. The finding that cyclin D1 is required for recruitment of G9a to target genes in chromatin and for H3K9 dimethylation, identifies a novel mechanism coordinating protein methylation. Published version This work was supported in part by R01CA70896, R01CA75503, and R01CA86072 (RGP). This project was funded in part from the Dr. Ralph and Marian C. Falk Medical Research Trust (RGP), a grant from the Breast Cancer Research Foundation (RGP), and a grant from the Pennsylvania Department of Health (RGP).

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that drives cell autonomous cell cycle progression and proliferation. Herein we show cyclin D1 abundance is increased >30-fold in the stromal fibroblasts of patients with invasive breast cancer, associated with poor outcome. Cyclin D1 transformed hTERT human fibroblast to a cancer-associated fibroblast phenotype. Stromal fibroblast expression of cyclin D1 (cyclin D1Stroma) in vivo, enhanced breast epithelial cancer tumor growth, restrained apoptosis, and increased autophagy. Cyclin D1Stroma had profound effects on the breast tumor microenvironment increasing the recruitment of F4/80+ and CD11b+ macrophages and increasing angiogenesis. Cyclin D1Stroma induced secretion of factors that promoted expansion of stem cells (breast stem-like cells, embryonic stem cells and bone marrow derived stem cells). Cyclin D1Stroma resulted in increased secretion of proinflammatory cytokines (CCL2, CCL7, CCL11, CXCL1, CXCL5, CXCL9, CXCL12), CSF (CSF1, GM-CSF1) and osteopontin (OPN) (30-fold). OPN was induced by cyclin D1 in fibroblasts, breast epithelial cells and in the murine transgenic mammary gland and OPN was sufficient to induce stem cell expansion. These results demonstrate that cyclin D1Stroma drives tumor microenvironment heterocellular signaling, promoting several key hallmarks of cancer. Published version

Both cell-autonomous and non–cell-autonomous factors contribute to tumor growth and metastasis of melanoma. The function of caveolin-1 (Cav1), a multifunctional scaffold protein known to modulate several biologic processes in both normal tissue and cancer, has been recently investigated in melanoma cancer cells, but its role in the melanoma microenvironment remains largely unexplored. Here, we show that orthotopic implantation of B16F10 melanoma cells in the skin of Cav1KO mice increases tumor growth, and co-injection of Cav1-deficient dermal fibroblasts with melanoma cells is sufficient to recapitulate the tumor phenotype observed in Cav1KO mice. Using indirect coculture experiments with fibroblasts and melanoma cells combined with cytokine analysis, we found that Cav1-deficient fibroblasts promoted the growth of melanoma cells via enhanced paracrine cytokine signaling. Specifically, Cav1-deficient fibroblasts displayed increased ShhN expression, which heterotypically enhanced the Shh signaling pathway in melanoma cells. In contrast to primary tumor growth, the ability of B16F10 melanoma cells to form lung metastases was significantly reduced in Cav1KO mice. This phenotype was associated mechanistically with the inability of melanoma cells to adhere to and to transmigrate through a monolayer of endothelial cells lacking Cav1. Together, our findings show that Cav1 may regulate different mechanisms during primary melanoma tumor growth and metastatic dissemination. Cancer Res; 72(9); 2262–74. ©2012 AACR.

The role of mammary epithelial cell (MEC) NF-κB in tumor progression in vivo is unknown, as murine NF-κB components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-κB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-κB, the importance of MEC NF-κB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-κB (IκBαSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-κB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-κB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that IκBαSR inhibited tumor stem cell pathways. IκBαSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-κB contributes to mammary tumorigenesis. As we show that NF-κB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-κB–dependent mammary tumorigenesis. Cancer Res; 70(24); 10464–73. ©2010 AACR.

The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in approximately 30% of human breast cancers. c-Jun, the first cellular proto-oncogene, is overexpressed in human breast cancer. However, the role of endogenous c-Jun in mammary tumor progression is unknown. Herein, transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-jun(f/f) transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration, invasion, and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal.

c-Jun mediates ROS production and apoptosis., c-jun, which is overexpressed in a number of human cancers encodes a critical component of the AP-1 complex. c-jun has been shown to either induce or inhibit cellular apoptosis. Germ line deletion of both c-jun alleles is embryonically lethal. To determine the role of the endogenous c-jun gene in apoptosis, we performed mammary epithelial cell–targeted somatic deletion using floxed c-jun (c-junf/f) conditional knockout mice. Laser capture microdissection demonstrated endogenous c-jun inhibits expression of apoptosis inducing genes and reactive oxygen species (ROS)-reducing genes (MnSOD, catalase). ROS have been implicated in apoptosis and undergo enzymatic elimination via MnSOD and CuZnSOD with further detoxification via catalase. c-jun–mediated survival was in part dependent on ROS production. c-jun–mediated repression of MnSOD and catalase occurred via mitochondrial complex I and NOX I. Collectively, these studies define a pivotal role of endogenous c-jun in promoting cell survival via maintaining mitochondrial integrity and expression of the key regulators of ROS production.

The (HER2/Neu) ErbB2 oncogene is commonly overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in transgenic mice. Nuclear factor (NF)-kappaB activity is increased in both human and murine breast tumors. The immune response to mammary tumorigenesis may regulate tumor progression. The role of endogenous mammary epithelial cell NF-kappaB had not previously been determined in immune-competent animals. Furthermore, the role of the NF-kappaB components, p50 and p65, in tumor growth was not known. Herein, the expression of a stabilized form of the NF-kappaB-inhibiting IkappaBalpha protein (IkappaBalphaSR) in breast tumor cell lines that express oncogenic ErbB2 inhibited DNA synthesis and growth in both two- and three-dimensional cultures. Either NF-kappaB inhibition or selective silencing of p50 or p65 led to a loss of contact-independent tumor growth in vitro. IkappaBalphaSR reversed the features of the oncogene-induced phenotype under three-dimensional growth conditions. The NF-kappaB blockade inhibited ErbB2-induced mammary tumor growth in both immune-competent and immune-deficient mice. These findings were associated with both reduced tumor microvascular density and a reduction in the amount of vascular endothelial growth factor. The expression of IkappaBalphaSR in breast cancer tumors inhibited angiogenesis. Thus, mammary epithelial cell NF-kappaB activity enhances ErbB2-mediated mammary tumorigenesis in vivo by promoting both growth and survival signaling via the promotion of tumor vasculogenesis.

Initially isolated as the dominant suppressor of the mutant epidermal growth factor receptor (ellipse), the Dachshund gene plays a key role in metazoan development regulating the Retinal Determination Gene Network. Herein, the DACH1 gene was expressed in normal prostate epithelial cells with reduced expression in human prostate cancer. DACH1 inhibited prostate cancer cellular DNA synthesis, growth in colony forming assays, and blocked contact-independent growth in soft agar assays. DACH1 inhibited androgen receptor (AR) activity, requiring a conserved DS Domain (Dachshund domain conserved with Ski/Sno) that bound NCoR/HDAC and was recruited to an androgen-responsive gene promoter. DACH1 inhibited ligand-dependent activity of AR mutations identified in patients with androgen-insensitive prostate cancer. The DS domain was sufficient for repression of the AR wild-type but failed to repress an AR acetylation site point mutant. These studies show a role for the Retinal Determination Gene Network in regulating cellular growth and signaling in prostate cancer. [Cancer Res 2009;69(8):3347–55]

The ErbB2 (Her2/neu epidermal growth receptor family) oncogene is overexpressed in 30% to 40% of human breast cancers. Cyclin D1 is the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma (pRb) tumor suppressor and is an essential downstream target of ErbB2-induced tumor growth. Herein, we demonstrate that ErbB2 induces the activity of the Notch signaling pathway. ErbB2 induction of DNA synthesis, contact-independent growth, and mammosphere induction required Notch1. ErbB2-induced cyclin D1 and cyclin D1 expression was suficient to induce Notch1 activity, and conversely, genetic deletion of Notch1 in mammary epithelial cells using foxed Notch (Notch(fl/fl)) mice demonstrated that cyclin D1 is induced by Notch1. Genetic deletion of cyclin D1 or small interfering RNA (siRNA) to cyclin D1-reduced Notch1 activity and reintroduction of cyclin D1 into cyclin D1-deficient cells restored Notch1 activity through the inhibition of Numb, an endogenous inhibitor of Notch1 activity. Thus, cyclin D1 functions downstream as a genetic target of Notch1, amplifies Notch1 activity by repressing Numb, and identifies a novel pathway by which ErbB2 induces Notch1 activity via the induction of cyclin D1.

Ascochyta blight (AB) caused by Ascochyta rabiei, is globally the most important foliar disease that limits the productivity of chickpea (Cicer arietinum L.). An intraspecific linkage map of cultivated chickpea was constructed using an F2 population derived from a cross between an AB susceptible parent ICC 4991 (Pb 7) and an AB resistant parent ICCV 04516. The resultant map consisted of 82 simple sequence repeat (SSR) markers and 2 expressed sequence tag (EST) markers covering 10 linkage groups, spanning a distance of 724.4 cM with an average marker density of 1 marker per 8.6 cM. Three quantitative trait loci (QTLs) were identified that contributed to resistance to an Indian isolate of AB, based on the seedling and adult plant reaction. QTL1 was mapped to LG3 linked to marker TR58 and explained 18.6% of the phenotypic variance (R2) for AB resistance at the adult plant stage. QTL2 and QTL3 were both mapped to LG4 close to four SSR markers and accounted for 7.7% and 9.3%, respectively, of the total phenotypic variance for AB resistance at seedling stage. The SSR markers which flanked the AB QTLs were validated in a half-sib population derived from the same resistant parent ICCV 04516. Markers TA146 and TR20, linked to QTL2 were shown to be significantly associated with AB resistance at the seedling stage in this half-sib population. The markers linked to these QTLs can be utilized in marker-assisted breeding for AB resistance in chickpea.

Oncogene-mediated signaling to the host environment induces a subset of cytokines and chemokines. TheDrosophila Dacgene promotes migration of the morphogenetic furrow during eye development. Expression of the cell-fate determination factorDachshund(DACH1) was lost in poor prognosis invasive breast cancer. Mouse embryo fibroblasts derived fromDach1−/−mice demonstrated endogenousDach1constitutively represses cellular migration. DACH1 inhibited cellular migration and invasion of oncogene (Ras, Myc, ErbB2, c-Raf)-transformed human breast epithelial cells. An unbiased proteomic analysis identified and immunoneutralizing antibody and reconstitution experiments demonstrated IL-8 is a critical target of DACH1 mediating breast cancer cellular migration and metastasisin vivo. DACH1 bound the endogenousIL-8promoter in ChIP assays and repressed theIL-8promoter through the AP-1 and NF-κB binding sites. Collectively, our data identify a pathway by which an endogenous cell-fate determination factor blocks oncogene-dependent tumor metastasis via a key heterotypic mediator.

The spread of metastatic tumors to different organs is associated with poor prognosis. The metastatic process requires migration and cellular invasion. The protooncogene c-jun encodes the founding member of the activator protein-1 family and is required for cellular proliferation and DNA synthesis in response to oncogenic signals and plays an essential role in chemical carcinogenesis. The role of c-Jun in cellular invasion remains to be defined. Genetic deletion of c-Jun in transgenic mice is embryonic lethal; therefore, transgenic mice encoding a c-Jun gene flanked by LoxP sites (c-junf/f) were used. c-jun gene deletion reduced c-Src expression, hyperactivated ROCK II signaling, and reduced cellular polarity, migration, and invasiveness. c-Jun increased c-Src mRNA abundance and c-Src promoter activity involving an AP-1 site in the c-Src promoter. Transduction of c-jun−/−cells with either c-Jun or c-Src retroviral expression systems restored the defective cellular migration of c-jun−/−cells. As c-Src is a critical component of pathways regulating proliferation, survival, and metastasis, the induction of c-Src abundance, by c-Jun, provides a novel mechanism of cooperative signaling in cellular invasion.

Cancer cells arise through sequential acquisition of mutations in tumor suppressors and oncogenes. c-Jun, a critical component of the AP-1 complex, is frequently overexpressed in diverse tumor types and has been implicated in promoting cellular proliferation, migration, and angiogenesis. Functional analysis of candidate genetic targets using germ line deletion in murine models can be compromised through compensatory mechanisms. As germ line deletion of c-jun induces embryonic lethality, somatic deletion of the c-jun gene was conducted using floxed c-jun (c-jun(f/f)) conditional knockout mice. c-jun-deleted cells showed increased cellular adhesion, stress fiber formation, and reduced cellular migration. The reduced migratory velocity and migratory directionality was rescued by either c-Jun reintroduction or addition of secreted factors from wild-type cells. An unbiased analysis of cytokines and growth factors, differentially expressed and showing loss of secretion upon c-jun deletion, identified stem cell factor (SCF) as a c-Jun target gene. Immunoneutralizing antibody to SCF reduced migration of wild-type cells. SCF addition rescued the defect in cellular adhesion, cellular velocity, directional migration, transwell migration, and cellular invasion of c-jun(-/-) cells. c-Jun induced SCF protein, mRNA, and promoter activity. Induction of the SCF promoter required the c-Jun DNA-binding domain. c-Jun bound to the SCF promoter in chromatin immunoprecipitation assays. Mutation of the c-Jun binding site abolished c-Jun-mediated induction of the SCF promoter. These studies demonstrate an essential role of c-Jun in cellular migration through induction of SCF.

Pyrazolopyrimidine and pyrimidopyrimidine derivatives have shown a wide range of biological activities such as acting as A1 adenosine receptors, kinase insert domain receptor (KDR), Rous sarcoma oncogene (Src), epidermal growth factor receptor (EGFR), antiproliferative, dihydrofolate reductase (DHFR), antimicrobial, antifungal, and lipid peroxidation. Because of this wide range of activities, we have synthesized pyrazolo[3,4‐d]pyrimidines and pyrimido[4,5‐d]pyrimidin‐4‐one derivatives.

Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27Kip1) is frequently reduced in human tumors, often correlating with poor prognosis. p27Kip1 functions as a haploinsufficient tumor suppressor; however, the mechanism by which one allele of p27Kip1 regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27Kip1 inhibits mammary tumor onset. Using the common background strain of FVB, p27Kip1 heterozygosity (p27+/−) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27Kip1 expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27+/− tumors showed that the loss of p27Kip1 induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/β-catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 and repressed by p27Kip1. Degradation of p27Kip1 involves an SCF-type E3 ubiquitin ligase, including Skp2. The Skp2 component of the SCFSKP2 complex that degrades p27Kip1 was increased in ErbB2 tumors correlating with earlier tumor onset. In both murine and human ErbB2-overexpressing breast cancers, p27Kip1 levels correlated inversely with Skp2. p27Kip1 haploinsufficiency activated Wnt/β-catenin/hedgehog signaling. Reintroduction of p27Kip1 inhibited β-catenin induction of Tcf-responsive genes (Siamosis, c-Myc, and Smad7). p27Kip1 is haploinsufficient for ErbB2 mammary tumor suppression in vivo and functions to repress collaborative oncogenic signals including Skp2 and Wnt/β-catenin signaling. (Cancer Res 2006; 66(17): 8529-41)

Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. Herein, cyclin D1 deficiency increased mitochondrial size and activity that was rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1), which induces nuclear-encoded mitochondrial genes, was repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis and mitochondrial function.

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.

A series of 21 compounds of trisubstituted pyrimidine derivatives have been synthesized and evaluated for their in vitro topoisomerase II inhibitory activity against filarial parasite Setaria cervi. Out of these, seven compounds (8, 11-14, 25 and 28) have shown 60-80% inhibition at 40 and 20 microg/mL concentration. Five compounds (12, 13, 14, 25 and 28) exhibited 70-80% inhibition at 10 microg/mL concentration and three compounds (13, 14 and 28) have shown 40-60% inhibition at 5 microg/mL concentration. All the above mentioned compounds have shown better topo II inhibitory activity than standard antifilarial drug (DEC) and enzyme topo II inhibitors (Novobiocin, Nalidixic acid).

Infection of high risk human papillomaviruses (HPVs) specifically the types 16 and 18 has been strongly implicated in the development of cervical cancer. The E6 oncoproteins of these high risk HPVs are known to bind and induce degradation of p53 tumour suppressor protein through the ubiquitin pathways. This degradation is controlled by a common polymorphism of the p53 gene encoding either a proline or an arginine at its codon 72 in exon 4. Recently, it has been demonstrated that the presence of homozygous arginine at codon 72 renders p53 about seven times more susceptible to E6-mediated proteolytic degradation as well as to cervical cancer than those with proline homozygotes or proline/arginine heterozygotes. In India, prevalence of HPV as well as cancers of the uterine cervix and the oral cavity are highest in the world. We have examined this allele-specific predisposition in cervical and oral cancer which is associated with HPV as well as in a non-HPV-linked cancer of the breast. We have carried out investigation in women comprising whole spectrum of cervical lesions with 128 HPV 16/18 positive and 35 HPV negative invasive cervical carcinomas and 34 cases of HPV (16/18) positive and 16 HPV negative cervical dysplasias (mild, moderate and severe) and 104 age-group-matched healthy women as controls. Additionally, we have analysed p53Arg-Pro polymorphism in 13 high risk HPV positive and 31 HPV negative oral cancers along with 20 normal controls and 77 breast cancers with 41 age-matched healthy controls. We observed more than 2 fold higher risk for homozygous arginine (χ2 = 6.3, df = 2, p = 0.04; OR = 2.3; 95% CI: 1.08–5.16) for HPV 16/18-positive cervical carcinomas when comparison was made only between HPV positive cervical cancers and normal controls but most interestingly, no significant association either in the frequency of homozygous arginine or proline alleles or their heterozygotes could be observed when all the three groups i.e. HPV-positive, HPV-negative cervical cancers and controls were considered simultaneously. No difference was also observed for either arginine or proline polymorphism between women with precancerous lesions of the uterine cervix carrying HPV 16/18 infection and controls. Similarly, increased risk of oral or breast cancer could not be correlated with the polymorphism of arginine/proline allele. Thus the interaction between HPV oncoproteins and the p53 gene polymorphism specifically, homozygous arginine at codon 72 appears to play no role in the development of either cervical or oral cancer and also it can not serve as a biomarker for early identification of cervical, oral or breast cancer.

Human papillomaviruses (HPVs) are major pathogens associated with the development of cancer of the uterine cervix, the most common malignant tumour of women worldwide. Reliable diagnosis of HPV infection, particularly the 'high-risk' types (16/18), may facilitate early identification of 'high-risk' populations for developing cervical cancer and may augment the sensitivity and specificity of primary cervical cancer screening programmes by complementing the conventional Pap test. A simple paper smear method has been developed for dry collection, transport and storage of cervical smears/scrapes at room temperature for subsequent detection of HPV DNA by PCR assay. Imprint biopsies, blood and fine-needle aspirates were also collected by this method. The cervical scrapes or other body fluids were smeared (within 0.5-1 cm diameter) and dried on to sterile small slides made of Whatman 3MM filter paper, and stored individually at room temperature or at 4 degrees C. A small piece (2-3 mm) of the paper smear was punched or cut out with a sterile surgical blade, boiled in an eppendorf tube containing 50 microl of distilled water for 5 min and used directly for PCR amplification. The quality and quantity of DNA derived from paper smears and the results of PCR amplifications for HPV type 16, BRCA1 and p53 genes were identical to those obtained from the same samples following collection in PBS, storage (-70 degrees C) and phenol-chloroform-based DNA extraction. DNA was stable in the paper smears for up to a year, whether stored at room temperature or at 4 degrees C. This method is simple, rapid and cost-effective, and can be effectively employed for large-scale population screening, especially for regions where the specimens are to be transported from distant places to the laboratory.

Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.

BACKGROUND Specific mutations of the p53 tumor suppressor gene in hepatocellular carcinoma (HCC) have been reported from several parts of the world, but to the authors' knowledge to date the status of this gene has not been studied in HCC patients in India, where HCC is one of the major cancers and the frequency of chronic hepatitis B virus (HBV) as well as hepatitis C virus (HCV) infection and exposure to dietary aflatoxin B1 is very high. The most frequent mutation of the p53 gene in HCC is an AGGArg to AGTSer missense mutation at codon 249 of exon 7. METHODS Liver biopsy specimens from 21 HCC patients and 10 healthy controls were obtained through surgery or by needle biopsy technique. Phenol-chloroform-extracted DNA specimens were employed for the detection of HBV infection and p53 gene mutations. Nucleotide mutations of exons 4–9 of the p53 gene were analyzed by polymerase chain reaction (PCR), single strand confirmation polymorphism, and direct sequencing. Third-generation sandwich enzyme-linked immunosorbent assay (ELISA) was used for the serologic detection of HBV and HCV infection. RESULTS Analysis of exons 4–9 of the p53 gene revealed only 3 mutations (3 of 21 specimens, 14.28%; 95% confidence interval, −0.7–29.3), 2 mutations at codon 249 showing GT transversions, and 1 mutation (4.7%) at codon 250 with a CT transition. The base substitutions at the third base of codon 249 resulted in a missense mutation leading to a change in amino acid from arginine to serine whereas at codon 250 it caused a change from proline to serine. Dot blot hybridization and PCR for HBV DNA from HCCs revealed 58.8% (10 of 17 specimens) and 90.47% (19 of 21 specimens), positivity, respectively. ELISA for hepatitis B virus surface antigen in serum showed a positivity of 71.42% (15 of 21 specimens), but there was only 40% positivity (8 of 20 specimens) for hepatitis B virus envelope antigen whereas 6 of 17 patients (35.29%) showed the presence of antibodies against hepatitis B virus envelope protein. No patient was found to be positive for the HCV antibody. CONCLUSIONS The very low frequency of p53 mutations and the extremely high frequency of HBV infection (> 90%) in HCC indicate that the mutations in the p53 gene frequently found in HCC reported from different endemic areas of the world may not play a direct role in the development of HCC in India. HBV infection and, possibly, exposure to the dietary aflatoxin B1 appear to play major roles in the molecular pathogenesis of HCC in India. Cancer 2000;88:1565–73. © 2000 American Cancer Society.

Improved clinical management of prostate cancer has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human prostate cancer. The TGF-β/BMP-SMAD4 signaling pathway, which induces epithelial–mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene–transformed prostate cancer cell lines. The in vivo cyclin D1–mediated molecular signature predicted poor outcome of recurrence-free survival for patients with prostate cancer (K-means HR, 3.75, P = 0.02) and demonstrated that endogenous cyclin D1 restrains TGF-β, Snail, Twist, and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In chromatin immunoprecipitation sequencing, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical-free recurrence in human prostate cancer. Cancer Res; 74(2); 508–19. ©2013 AACR.

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture microdissected c-jun−/− mammary epithelium, showed that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays showed that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directly attenuates RNA splicing efficiency, which may be of broad biologic importance as alternative splicing plays an important role in both cancer development and therapy resistance. Cancer Res; 72(4); 1023–34. ©2011 AACR.

Tumor progression involves the acquisition of invasiveness through a basement membrane. The c-jun proto-oncogene is overexpressed in human tumors and has been identified at the leading edge of human breast tumors. TGF-β plays a bifunctional role in tumorigenesis and cellular migration. Although c-Jun and the activator protein 1 (AP-1) complex have been implicated in human cancer, the molecular mechanisms governing cellular migration via c-Jun and the role of c-Jun in TGF-β signaling remains poorly understood. Here, we analyze TGF-β mediated cellular migration in mouse embryo fibroblasts using floxed c-jun transgenic mice. We compared the c-jun wild type with the c-jun knockout cells through the use of Cre recombinase. Herein, TGF-β stimulated cellular migration and intracellular calcium release requiring endogenous c-Jun. TGF-β mediated Ca(2+) release was independent of extracellular calcium and was suppressed by both U73122 and neomycin, pharmacological inhibitors of the breakdown of PIP(2) into IP(3). Unlike TGF-β-mediated Ca(2+) release, which was c-Jun dependent, ATP mediated Ca(2+) release was c-Jun independent. These studies identify a novel pathway by which TGF-β regulates cellular migration and Ca(2+) release via endogenous c-Jun.

microRNAs are thought to regulate tumor progression and invasion via direct interaction with target genes within cells. Here the microRNA17/20 cluster is shown to govern cellular migration and invasion of nearby cells via heterotypic secreted signals. microRNA17/20 abundance is reduced in highly invasive breast cancer cell lines and node-positive breast cancer specimens. Cell-conditioned medium from microRNA17/20–overexpressing noninvasive breast cancer cell MCF7 was sufficient to inhibit MDA-MB-231 cell migration and invasion through inhibiting secretion of a subset of cytokines, and suppressing plasminogen activation via inhibition of the secreted plasminogen activators (cytokeratin 8 and α-enolase). microRNA17/20 directly repressed IL-8 by targeting its 3′ UTR, and inhibited cytokeratin 8 via the cell cycle control protein cyclin D1. At variance with prior studies, these results demonstrated a unique mechanism of how the altered microRNA17/20 expression regulates cellular secretion and tumor microenvironment to control migration and invasion of neighboring cells in breast cancer. These findings not only reveal an antiinvasive function of miR-17/20 in breast cancer, but also identify a heterotypic secreted signal that mediates the microRNA regulation of tumor metastasis.

p21 CIP1/WAF1 is a downstream effector of tumor suppressors and functions as a cyclin-dependent kinase inhibitor to block cellular proliferation. Breast tumors may derive from self-renewing tumor-initiating cells (BT-ICs), which contribute to tumor progression, recurrence, and therapy resistance. The role of p21 CIP1 in regulating features of tumor stem cells in vivo is unknown. Herein, deletion of p21 CIP1 , which enhanced the rate of tumorigenesis induced by mammary-targeted Ha-Ras or c-Myc, enhanced gene expression profiles and immunohistochemical features of epithelial mesenchymal transition (EMT) and putative cancer stem cells in vivo. Silencing of p21 CIP1 enhanced, and expression of p21 CIP1 repressed, features of EMT in transformed immortal human MEC lines. p21 CIP1 attenuated oncogene-induced BT-IC and mammosphere formation. Thus, the in vitro cell culture assays reflect the changes observed in vivo in transgenic mice. These findings establish a link between the loss of p21 CIP1 and the acquisition of breast cancer EMT and stem cell properties in vivo.