Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening.

BACKGROUND: MYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers. METHODS: A pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified that PRKDC was synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knocked down or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined mRNA expression levels and cell viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Statistical significance of differences among data sets were determined using unpaired t test (Mann-Whitney test) or ANOVA. RESULTS: Inhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels. CONCLUSIONS: Our data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair.

Membrane metalloendopeptidase suppresses prostate carcinogenesis by attenuating effects of gastrin-releasing peptide on stem/progenitor cells.

Aberrant neuroendocrine signaling is frequent yet poorly understood feature of prostate cancers. Membrane metalloendopeptidase (MME) is responsible for the catalytic inactivation of neuropeptide substrates, and is downregulated in nearly 50% of prostate cancers. However its role in prostate carcinogenesis, including formation of castration-resistant prostate carcinomas, remains uncertain. Here we report that MME cooperates with PTEN in suppression of carcinogenesis by controlling activities of prostate stem/progenitor cells. Lack of MME and PTEN results in development of adenocarcinomas characterized by propensity for vascular invasion and formation of proliferative neuroendocrine clusters after castration. Effects of MME on prostate stem/progenitor cells depend on its catalytic activity and can be recapitulated by addition of the MME substrate, gastrin-releasing peptide (GRP). Knockdown or inhibition of GRP receptor (GRPR) abrogate effects of MME deficiency and delay growth of human prostate cancer xenografts by reducing the number of cancer-propagating cells. In sum, our study provides a definitive proof of tumor-suppressive role of MME, links GRP/GRPR signaling to the control of prostate stem/progenitor cells, and shows how dysregulation of such signaling may promote formation of castration-resistant prostate carcinomas. It also identifies GRPR as a valuable target for therapies aimed at eradication of cancer-propagating cells in prostate cancers with MME downregulation.

Prostate cancer associated with p53 and Rb deficiency arises from the stem/progenitor cell-enriched proximal region of prostatic ducts.

Recently, we have shown that prostate epithelium-specific deficiency for p53 and Rb tumor suppressors leads to metastatic cancer, exhibiting features of both luminal and neuroendocrine differentiation. Using stage-by-stage evaluation of carcinogenesis in this model, we report that all malignant neoplasms arise from the proximal region of the prostatic ducts, the compartment highly enriched for prostatic stem/progenitor cells. In close similarity to reported properties of prostatic stem cells, the cells of the earliest neoplastic lesions express stem cell marker stem cell antigen 1 and are not sensitive to androgen withdrawal. Like a subset of normal cells located in the proximal region of prostatic ducts, the early neoplastic cells coexpress luminal epithelium markers cytokeratin 8, androgen receptor, and neuroendocrine markers synaptophysin and chromogranin A. Inactivation of p53 and Rb also takes place in the lineage-committed transit-amplifying and/or differentiated cells of the distal region of the prostatic ducts. However, the resulting prostatic intraepithelial neoplasms never progress to carcinoma by the time of mouse death. Interestingly, in an ectopic transplantation assay, early mutant cells derived from either region of the prostatic ducts are capable of forming neoplasms within 3 months. These findings indicate that p53 and Rb are critically important for the regulation of the prostatic stem cell compartment, the transformation in which may lead to particularly aggressive cancers in the context of microenvironment.

Mouse models of prostate adenocarcinoma with the capacity to monitor spontaneous carcinogenesis by bioluminescence or fluorescence.

The application of Cre/loxP technology has resulted in a new generation of conditional mouse models of prostate cancer. Here, we describe the improvement of the conditional Pten deletion model of prostate adenocarcinoma by combining it with either a conditional luciferase or enhanced green fluorescent protein reporter line. In these models, the recombination mechanism that inactivates the Pten alleles also activates the reporter gene. In the luciferase reporter model, the growth of the primary cancer can be followed noninvasively by bioluminescence imaging (BLI). Surgical castration of tumor-bearing animals leads to a reduced bioluminescence signal corresponding to tumor regression that is verified at necropsy. When castrated animals are maintained, the emergence of androgen depletion-independent cancer is detected using BLI at times varying from 7 to 28 weeks postcastration. The ability to monitor growth, regression, or relapse of the tumor with the use of BLI lead to the collection of tumors at different stages of development. By comparing the distribution of phenotypically distinct populations of epithelial cells in cancer tissues, we noted that the degree of hyperplasia of cells with neuroendocrine differentiation significantly increases in the recurrent cancer relative to the primary cancer, a characteristic which may parallel the appearance of a neuroendocrine phenotype in human androgen depletion-independent cancer. The enhanced green fluorescent protein model, at necropsy, can provide an opportunity to locate or assess tumor volume or to isolate enriched populations of cancer cells from tumor tissues via fluorescence-based technologies. These refined models should be useful in the elucidation of mechanisms of prostate cancer progression, and for the development of approaches to preclinical intervention.

Cell lineage-specific interactions between Men1 and Rb in neuroendocrine neoplasia.

Inactivation of multiple endocrine neoplasia (MEN) type 1 gene (Men1) results in development of multiple endocrine tumors in Men1(+/-) mice and in humans. Intriguingly, loss of the wild-type retinoblastoma 1 (Rb) gene also leads to MEN-like phenotype in Rb(+/-) mice. To evaluate potential genetic interactions between these genes, we prepared and characterized Men1(+/-)Rb(+/-) compound mice in parallel with their parental genotypes. Men1 and Rb did not cooperate in tumor suppression, as demonstrated by comparable survival rates of Rb(+/-) and Men1(+/-)Rb(+/-) mice, absence of tumor growth acceleration and lack of novel neoplasms. Notably, the loss of the remaining copy of the wild-type Men1 and Rb was mutually exclusive in all tumors of Men1(+/-)Rb(+/-) mice, including pituitary anterior lobe and adrenal medulla neoplasms shared by Rb- and Men1-deficient phenotypes. Down-regulation of Men1 targets p18 and p27 and increased presence of phosphorylated-Rb were observed in Men1-deficient pheochromocytomas of Men1(+/-)Rb(+/-) and Men1(+/-) mice. At the same time, the RNA interference (RNAi) knock-down of Men1 mRNA resulted in increased apoptosis of Rb-deficient medullary thyroid carcinoma cells. These results demonstrate that, depending on cell lineage context, combined Men1 and Rb deficiency may be either redundant or detrimental to neoplastic growth. Identification of cell lineage-specific interactions between Men1 and Rb may have important implications for development of rationally designed therapeutic approaches.

Core-shell silica nanoparticles as fluorescent labels for nanomedicine.

Progress in biomedical imaging depends on the development of probes that combine low toxicity with high sensitivity, resolution, and stability. Toward that end, a new class of highly fluorescent core-shell silica nanoparticles with narrow size distributions and enhanced photostability, known as C dots, provide an appealing alternative to quantum dots. Here, C dots are evaluated with a particular emphasis on in-vivo applications in cancer biology. It is established that C dots are nontoxic at biologically relevant concentrations, and can be used in a broad range of imaging applications including intravital visualization of capillaries and macrophages, sentinel lymph node mapping, and peptide-mediated multicolor cell labeling for real-time imaging of tumor metastasis and tracking of injected bone marrow cells in mice. These results demonstrate that fluorescent core-shell silica nanoparticles represent a powerful novel imaging tool within the emerging field of nanomedicine.

Suppression of melanotroph carcinogenesis leads to accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas in Rb+/- mice.

Mice with a single copy of the retinoblastoma gene (Rb(+/-)) develop a syndrome of multiple neuroendocrine neoplasia. They usually succumb to fast-growing, Rb-deficient melanotroph tumors of the pituitary intermediate lobe, which are extremely rare in humans. Thus, full assessment of Rb role in other, more relevant to human pathology, neoplasms is complicated. To prevent melanotroph neoplasia while preserving spontaneous carcinogenesis in other types of cells, we have prepared transgenic mice in which 770-bp fragment of pro-opiomelanocortin promoter directs expression of the human RB gene to melanotrophs (TgPOMC-RB). In three independent lines, transgenic mice crossed to Rb(+/-) background are devoid of melanotroph tumors but develop the usual spectrum of other neoplasms. Interestingly, abrogation of melanotroph carcinogenesis results in accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas. A combination of immunologic tests, cell culture studies, and tumorigenicity assays indicates that alpha-melanocyte-stimulating hormone, which is overproduced by melanotroph tumors, attenuates neoplastic progression by decreasing cell proliferation and inducing apoptosis. Taken together, we show that cell lineage-specific complementation of Rb function can be successfully used for refining available models of stochastic carcinogenesis and identify alpha-melanocyte-stimulating hormone as a potential attenuating factor during progression of neuroendocrine neoplasms.

A novel full-length gene of human ribosomal protein L14.22 related to human glioma.

BACKGROUND: This study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene. METHODS: Total RNA was extracted form human glioma and normal brain tissue, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08 cone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression. RESULTS: Fifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08 clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBank with the accession number of AF329277. After expression in E. coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel. CONCLUSIONS: cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human ribosomal protein 13.22 may be correlated with the development of human glioma.

Cloning, expression and characterisation of a human Nudix hydrolase specific for adenosine 5'-diphosphoribose (ADP-ribose).

The human NUDT9 gene has been mapped to 4q22 and shown to give rise to two alternatively spliced mRNAs, NUDT9alpha and NUDT9beta, that encode a member of the Nudix hydrolase family. Both transcripts were readily detected in heart and skeletal muscle and also in liver, kidney and pancreas. NUDT9alpha protein was expressed in Escherichia coli and shown specifically to hydrolyse ADP-ribose and IDP-ribose to the corresponding nucleoside 5'-monophosphates and ribose 5-phosphate. No other nucleotide substrates were hydrolysed significantly. NUDT9alpha was inhibited by fluoride and by N-acetyl-p-benzoquinoneimine and had K(m) and kcat values of 180 microM and 8 s(-1) respectively with ADP-ribose as substrate. The full-length 39.1 kDa NUDT9alpha has a potential mitochondrial leader sequence, which would give rise to a mature 34.2 kDa mitochondrial protein. Apart from the high K(m) value, the properties of NUDT9alpha are close to those of the known mammalian 40 kDa cytoplasmic ADPRibase-1 and 35 kDa mitochondrial ADPRibase-m. However, any relationship between the NUDT9 species and the previously reported ADPRibases remains to be established.

cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene.

BACKGROUND: This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. METHODS: Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. RESULTS: Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b). CONCLUSIONS: cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

Cloning and characterization of ARHGAP12, a novel human rhoGAP gene.

Rho GTPase activating proteins (GAPs) stimulate the intrinsic GTP hydrolysis activity of Rho family proteins. Here we report the cloning of two splice variants of a novel gene named ARHGAP12 (access number), which has an ORF of 2541bp. Profilescan search result showed that its putative protein contains five domains: rhoGAP, SH3, PH and two WW domains. ARHGAP12 is located in chromosome 10pter-cen and consists of 20 exons according to the Blastn result against high throughput genomic sequences (htgs). Reverse transcription PCR and Northern blot indicates that it ubiquitously expresses in human tissues as well as tumor cell lines, suggesting its basic roles in cells.

Cloning and characterization of a novel human transcription factor AP-2 beta like gene (TFAP2BL1).

The AP-2 transcription factor has been shown to play an important role in development, morphogenesis, apoptosis, cell-cycle control and has also been implicated in mammary oncogenesis. Here we report the cloning and characterization of a novel human transcription factor AP-2 like gene (TFAP2BL1), which is located on human chromosome 6p12.1-21.1. The TFAP2BL1 cDNA is 2076 base pairs in length, encoding a 452-amino acid polypeptide related to human Ap-2protein. TFAP2BL1 gene has significantly high homology to transcription factor AP-2 gene of human, mouse, chicken, sheep, fruit fly, and C. elegans at amino acid level. RT-PCR analysis shows its relatively high expression level in adult thymus, prostate, small intestine, skeletal muscle, placenta, brain, and testis tissues.

Identification and characterization of AGTRAP, a human homolog of murine Angiotensin II Receptor-Associated Protein (Agtrap).

Several classes of cytoplasmic proteins have been found to interact specifically with the carboxyl-terminal cytoplasmic region of the angiotensin II type 1 (AT(1)) receptor to regulate different aspects of AT(1) receptor physiology. The murine Angiotensin II Receptor-Associated Protein (Agtrap) is a new member of them. We have recently cloned a new human gene cDNA that codes for a homolog of the murine Agtrap protein from a human fetal brain cDNA library. The deduced polypeptide product of the cDNA is 22 kDa in size, and its DNA and amino acid sequences are 85 and 77% identical to those of the mouse Agtrap gene, respectively. Hence we have named it the human Angiotensin II Receptor-Associated Protein (AGTRAP) gene. The mRNA of AGTRAP was most abundantly expressed in kidney, heart, pancreas and thyroid. Using the yeast two-hybrid screening of a human fetal brain cDNA library, we have identified a new interaction partner of the human AGTRAP protein, RACK1 (Receptor of Activated Protein C Kinase). The AGTRAP-RACK1 interaction was confirmed by GST fusion protein pull-down assays, co-immunoprecipitation and surface plasmon resonance. We suggest that the AGTRAP-RACK1 interaction may help to recruit signaling complex to the AT(1) receptor to affect AT(1) receptor signaling.

[Tumor relevance analysis of a highly conserved gene by using gene microarray hybridization].

Preliminary function research of a highly conserved human gene,which was cloned from human fetal cDNA library during large-scale cDNA sequencing,is illustrated in this article. Bioinformatics analysis indicates that this gene is highly conserved in human, mouse, fruit fly, thaliana and fission yeast. Other bioinformatics analysis implies its relevance with tumors. RT-PCR analysis shows its wide-ranging expression patterns. Its expression in 16 cancer cases (including 7 liver cancer cases, 5 pancreas cancer cases, 2 larynx cancer cases and 2 lung cancer cases) is studied by using gene microarray analysis. The result shows its relevance with tumors and implies it may have different status in different classification of tumors.

Detection and organ-specific ablation of neuroendocrine cells by synaptophysin locus-based BAC cassette in transgenic mice.

The role of cells of the diffuse neuroendocrine system in development and maintenance of individual organs and tissues remains poorly understood. Here we identify a regulatory region sufficient for accurate in vivo expression of synaptophysin (SYP), a common marker of neuroendocrine differentiation, and report generation of Tg(Syp-EGFP(loxP)-DTA)147(Ayn) (SypELDTA) mice suitable for flexible organ-specific ablation of neuroendocrine cells. These mice express EGFP and diphtheria toxin fragment A (DTA) in SYP positive cells before and after Cre-loxP mediated recombination, respectively. As a proof of principle, we have crossed SypELDTA mice with EIIA-Cre and PB-Cre4 mice. EIIA-Cre mice express Cre recombinase in a broad range of tissues, while PB-Cre4 mice specifically express Cre recombinase in the prostate epithelium. Double transgenic EIIA-Cre; SypELDTA embryos exhibited massive cell death in SYP positive cells. At the same time, PB-Cre4; SypELDTA mice showed a substantial decrease in the number of neuroendocrine cells and associated prostate hypotrophy. As no increase in cell death and/or Cre-loxP mediated recombination was observed in non-neuroendocrine epithelium cells, these results suggest that neuroendocrine cells play an important role in prostate development. High cell type specificity of Syp locus-based cassette and versatility of generated mouse model should assure applicability of these resources to studies of neuroendocrine cell functions in various tissues and organs.

MET-dependent cancer invasion may be preprogrammed by early alterations of p53-regulated feedforward loop and triggered by stromal cell-derived HGF.

MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR-34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.

Selenite triggers rapid transcriptional activation of p53, and p53-mediated apoptosis in prostate cancer cells: Implication for the treatment of early-stage prostate cancer.

Supra-nutritional selenium supplementation has emerged as an attractive new approach to intervene in a range of human cancers, in particular prostate cancer. However, scanty information is currently available on molecular mechanisms underlying selenium's anticancer action. The tumor suppressor p53 plays an important role in preventing transformation by transcriptional regulation of a range of genes that are involved in vital cell functions such as DNA repair, cell cycle arrest, and induction of apoptosis. Here we report that incubation of LNCaP human prostate cancer cells (p53 +/+) with a natural form of selenium triggers rapid transcriptional activation of p53, and up-regulation of the expression of p53-target genes as well as induction of miR-34 class of microRNAs. Moreover, blocking p53 function by transfection of cells with a dominant-negative, mutated p53 gene, or by siRNA, significantly reduced selenium-induced expression of p53-target genes and induction of apoptosis. Since majority of the early-stage human prostate cancers bear functional p53 gene (p53+/+), our findings indicate that the anticancer action of selenium may involve transactivation of p53 as a potential mechanism, and suggest that selenite may be useful not only for prevention but also for treatment of human prostate cancer.

Pro-neural transcription factors as cancer markers.

BACKGROUND: The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours. RESULTS: Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers. CONCLUSION: By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.

Synergy of p53 and Rb deficiency in a conditional mouse model for metastatic prostate cancer.

Pathways mediated by p53 and Rb are frequently altered in aggressive human cancers, including prostate carcinoma. To test directly the roles of p53 and Rb in prostate carcinogenesis, we have conditionally inactivated these genes in the prostate epithelium of the mouse. Inactivation of either p53 or Rb leads to prostatic intraepithelial neoplasia developing from the luminal epithelium by 600 days of age. In contrast, inactivation of both genes results in rapidly developing (median survival, 226 days) carcinomas showing both luminal epithelial and neuroendocrine differentiation. The resulting neoplasms are highly metastatic, resistant to androgen depletion from the early stage of development, and marked with multiple gene expression signatures commonly found in human prostate carcinomas. Interestingly, gains at 4qC3 and 4qD2.2 and loss at 14qA2-qD2 have been consistently found by comparative genomic hybridization. These loci contain such human cancer-related genes as Nfib, L-myc, and Nkx3.1, respectively. Our studies show a critical role for p53 and Rb deficiency in prostate carcinogenesis and identify likely secondary genetic alterations. The new genetically defined model should be particularly valuable for providing new molecular insights into the pathogenesis of human prostate cancer.