"Ectopic Expression of Germline Genes Drives Malignant Brain Tumor Growth in Drosophila".

Ana Janic ¹, Leire Mendizabal ^1,*, Salud Llamazares ¹, David Rossell ², and Cayetano Gonzalez ^{1, 3,} †

¹ Cell Division Group, Institute for Research in Biomedicine (IRB-Barcelona), PCB, c/Baldiri Reixac 10-12, Barcelona, Spain.
² Bioinformatics and Biostatistics Unit, IRB-Barcelona, PCB, c/Baldiri Reixac 10-12, Barcelona, Spain.
³ Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, Barcelona, Spain.

 †To whom correspondence should be addressed. E-mail: gonzalez@irbbarcelona.org

* Present address: Genomics Core Facility, Vall d'Hebrón Institute of Oncology, Passeig Vall d'Hebron 119, 08035 Barcelona, Spain.

Received for publication 22 July 2010.  Accepted for publication 9 November 2010.

NetworkEditors' Perspectives: "Embryoma gene netwoks from germ cells within adult neoplasms".
Abstract:
Introduction:
Methods:
Results:
   Fig. 1: Gene expression profile of mbt tumors.
   Fig. 2: Ectopic expression of germline proteins in l(3)mbt^ts1
    Fig. 3: The role of ectopically expressed germline genes in mbt tumor growth.
Supporting online material:
   Supplemental Material and Methods:
   Fig.S1: Genome-wide gene expression profiles of mbt tumors and control brains.
   Table S1. Gene expression values.
   Table S2. Relative expression levels of the genes that are most significantly dysregulated in mbt tumors.
   Table S3. Average expression levels of the genes that are most significantly dysregulated in mbt tumors.
   Table S4. Germline genes overexpressed in mbt tumors.
   Table S5. miRNAs and piRNAs expressed in mbt tumors.
   Supplemental References:
References and Notes:
Citied by:
Additional References:
Conclusions from: Euchromatin, Embryomas, Entropy, Enhancers, EMT,
Further Topics:

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. Here we show that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. Our results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species.

The Drosophila tumor-suppressor gene lethal (3) malignant brain tumor [l(3)mbt] was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain (1). Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype (2). L(3)mbt’s closest homologs, DrosophilaScm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins (3). L3MBTL1, the human homolog of Drosophila L(3)MBT (3), is a transcriptional repressor (4) that is found in a complex with core histones, heterochromatin protein 1g (HP1_g), and RB (Retinoblastoma protein) and can compact nucleosomes (5). Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex (6). Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression (7). These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (3, 7).

To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), we carried out genome-wide gene expression profiling of l(3)mbtE2 and l(3)mbtts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). We also analyzed l(3)mbtts1 tumors at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white1118 (w1118), l(3)mbtE2, or l(3)mbtts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, we also profiled larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins) (8).

Hierarchical clustering plots of these data (table S1) reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbtts1 tumors, respectively (fig. S1). From these data, we identified 151 genes that were either overexpressed (n = 125) or underexpressed (n = 26) in all three larval mbt tumor types compared to all three controls (table S2). From this list, we removed those genes that were also up- (n = 23) or down-regulated (n = 14) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. We refer to the expression levels of the remaining 102 up-regulated genes as the mbt signature (MBTS) (table S3). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat (Fig. 1A and table S3). Individual MBTS genes, however, are also up-regulated in some of these tumors.

Fig. 1: Gene expression profile of mbt tumors.

Fig. 1: Gene expression profile of mbt tumors.

(A) Heatmap of expression levels of the genes that are most significantly up-regulated in larval brain mbt tumors (mbt tumor signature, MBTS). Samples include wild-type larval brains, larval mbt tumors, and different types of larval brain malignant neoplasms in the 1st, 5th, and 10th rounds of allograft culture (T1, T5, and T10, respectively).

(B) MBTS genes with known germline functions.

The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line (Fig. 1B and table S4A). For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation (9). The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific gTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development (10–15). Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses (16, 17). Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality (18), and nos is required during nervous system development (19).

Driven by the high percentage of MBTS genes that have germline functions, we looked for other germline-related genes that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors (table S4B). Among these we found the genes that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively (11, 13). The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (16, 17).

To determine whether the mRNAs that we found ectopically expressed in mbt tumors are translated, we checked for protein expression with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development (20), we included it in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, we confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors (Fig. 2A). Immunofluorescence studies also revealed the ectopic expression in l(3)mbtts1 brains raised at 29°C of C(3)G, SQU, and VASA (Fig. 2B). These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, we have not been able to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated.

Fig. 2: Ectopic expression of germline proteins in l(3)mbt^ts1

Fig. 2: Ectopic expression of germline proteins in l(3)mbt^ts1

(A) Western blot. PIWI, AUB, and VASA are ectopically expressed in l(3)mbtts1 brain tumors. aTUB is used as a loading control.

(B) Immunofluorescence. VASA, SQU, and C(3)G are overexpressed in l(3)mbtts1 brains raised at 29°C. Low-magnification views (left) reveal VASA staining concentrated in the outer proliferative center (OPC) and in undifferentiated cells of the central brain (CB) (scale bar, 50 mm). High-magnification views (middle and right) show that SQU and C(3)G localize in the cytoplasm and on condensed chromatin, respectively (scale bar, 10 mm). Brains were counterstained with DAPI (4',6'-diamidino-2-phenylindole) (DNA) and antibodies against the neuroblast marker MIRA.

Prompted by the expression in l(3)mbtts1 brains of several genes involved in the biogenesis and regulation of piRNAs, we sequenced 23- to 30-nucleotide RNAs from l(3)mbtts1 larval brain tumors and from wild-type brains and ovaries. We found 117 known piRNAs and microRNAs (miRNAs) in l(3)mbtts1 larval brain tumor samples (table S5). Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors.

We do not know which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component (6), has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells (7). In addition, we do not have an estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (21–23).

We then determined the extent to which ectopic expression of germline genes contributes to mbt tumor growth. To this end, we first quantified larval brain growth in individuals that were mutant for l(3)mbtts1 alone, or double mutant for l(3)mbtts1 and one of several of the germline genes that are ectopically expressed in mbt tumors (Fig. 3). Measured as the total amount of protein, the average brain size in l(3)mbtts1 (21 ± 6 mg of protein per brain, n = 5) is about seven times as large (P < 0.0001) as that in control w¹¹¹⁸ larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbtts1 larvae that are also mutant for either piwi (P < 0.0001), vasa (P < 0.0001), aub (P = 0.0003), or nos (P = 0.001) (Fig. 3). The loss of piwi does not prevent brain overgrowth in brat k06028 mutant larvae (P = 0.72). We then quantified tumor growth after allograft in adult flies (Fig. 3). The frequency with which l(3)mbtts1 homozygous larval brain tissue develops tumors in this assay (70%, n = 67) is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt (P = 0.03), but it is markedly reduced by the additional loss of piwi (P < 0.0001), vasa (P < 0.0001), aub (P = 0.0002), or nos (P < 0.0001). The frequency of brat k06028 tumor formation (80%, n = 10) is not affected by the loss of piwi (73%, n = 15) or nos (86%, n = 7, P = 1). These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth.

Fig. 3: The role of ectopically expressed germline genes in mbt tumor growth.

Fig. 3: The role of ectopically expressed germline genes in mbt tumor growth.

Brain micrographs were taken from larvae of the corresponding genotypes raised at 29°C (scale bars, 100 mm). Larval brain size is shown as mean ± SD (in micrograms) of protein per brain (n = number of brains). Adult fly micrographs were taken 10 days after implantation of green fluorescent protein (GFP)–labeled larval brain tissue. In the absence of tumor growth, GFP signal is either undetectable or is localized to a very small piece of green tissue that is about the size of the implant (arrows). Tumor growth was quantified as the percentage (%) of n hosts in which the implanted tissue (green) grew over the entire abdomen of the host. P-values refer to the difference between each double-mutant combination and l(3)mbtts1, or between piwi1 bratK06028 and bratk06028.

A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains (20, 24, 25), has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells (24, 25). Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas (26, 27). These are known as cancer-testis (CT) genes or cancer-germline (CG) genes (28). A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (29, 30).

Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation (28), but their actual role in cancer remains unknown. Our results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi (31, 32), NANOS1/nanos (33), and SYCP1 /c(3)G (34). The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer.

Supporting Online Material:

http://www.sciencemag.org/cgi/content/full/330/6012/1824/DC1
 

Contents
Material and Methods……... p. 3
Fig. S1……………………… p. 5
Table S1…………………… p. 6
Table S2…………………… p. 158
Table S3…………………… p. 160
Table S4…………………… p. 162
Table S5…………………… p. 163
References………………… p. 167

Fly strains and generation of double mutant lines

The following mutant alleles were used in this study: l(3)mbtE2 (S1), l(3)mbtts1 (S2), piwi1 ( S3), zpgz-2533 (
S4), Pxtf01000 ( S5), AGO3t3 ( S6), vasa PH165( S7), aubQC42 ( S8), nos17( S9), Df(3R)D1-FX1 (S10),
Df(2L)A267 ( S7), brat k06028 ( S11), pins89 and pins62 ( S12), pros17 ( S13), miraZZ176 ( S14), and lgl4 ( S15).
Additional information on the function of these genes can be found in Flybase (http://www.flybase.org).
MARCM pros17 and miraZZ176 clones were generated by FLP/FRT mediated mitotic recombination ( S16).
Double mutant combinations were obtained using standard genetic techniques.

RNA isolation

Total RNA was prepared using Trizol (Invitrogen) following the manufacturer’s instructions. Purity and
integrity of the purified RNA was assessed on the Agilent Bioanalyzer 2100. Concentration was determined
with a Nanodrop ND-1000 Spectrophotometer.

Microarray Expression Profiling

Labeling and hybridization of the samples to Affymetrix GeneChip® Drosophila Genome 2.0 Array were
performed according to the manufacturer’s instructions (http://www.affymetrix.com). Probe set based gene
expression measurements were generated from Affymetrix image files (“.CEL” files) using quantile ( S17)
normalization and GCRMA summarization ( S18), as implemented in the gcrma function in the
Bioconductor gcrma package. A Principal Components Analysis revealed that microarrays hybridized in
two different laboratories presented technical biases. We clustered samples via hierarchical clustering with
Euclidean distance and complete linkage. We removed those biases by computing relative expression
levels, i.e. subtracting the average expression of the controls within each laboratory. For each sample we
classified genes into expression patterns using the GaGa model( S19). We defined expression patterns to
capture the first generation in which the expression levels were significantly different from those in the
control samples, and an additional pattern for non-differentially expressed genes. Genes were selected by
setting the Bayesian FDR at 0.05 and requiring an absolute fold change vs. control sample larger than 2.

Deep sequencing analysis

The small RNA fraction was isolated from total RNA (~10µg) using the mirVana small RNA isolation kit
(Ambion). 23 – 30 nucleotides RNAs were obtained by passive elution of the RNA fraction from
denaturing 15% polyacrylamide (PAA) gels. These were then poly(A)-tailed using poly(A) polymerase,
followed by ligation of a RNA adapter to the 5´-phosphate of the RNA. RNA fragments with adapters were
reverse transcribed and the resulting cDNAs were amplified to 20-30ng/µl in 21 PCR cycles. PCR products
were analyzed on 6% PAA gels and the main bands in the 110-125 nucleotide range were isolated. The
purified DNA fragments were analyzed on the Illumina/Solexa Genome Analyser II. Image analysis and
base calling were performed with the Illumina Pipeline software as available on April 2009. Before
mapping the reads to the genome, the poly-A tail was clipped and reads shorter than 5 nucleotides were
excluded. Reads were mapped to the Drosophila melanogaster genome (April 2006 release) using the
Bowtie software version 0.9.9.1 ( S20). Reads mapping to several places in the genome were excluded from
all analyses. The coverage for each sample, i.e. the number of reads mapped to each position in the
reference genome, was computed with the Bioconductor ShortRead package version 1.0.7 ( S21, S22).
Chromosomal positions covered by at least 10 reads were selected. Read counts in these regions were
compared between samples with a Poisson exact test. Chromosomal loci that had adjusted Benjamini-
Yekutieli p-value ( S23) below 0.05 were selected as enriched. Enriched chromosomal loci less than 10
bases apart were grouped into enriched regions. The average coverage in the significantly enriched regions
is shown in Table S5. Significantly enriched regions were annotated using the GSM231091 ( S24) and
GSE6734 ( S25) piRNA libraries available at Gene Expression Omnibus ( S26). Enriched regions that
overlap with regions referred to in piRNA databases were annotated according to the corresponding
database. Additionally, miRNAs were annotated according to the miRBase database (June, 2009).

Western blots

Protein extracts from larval brains, ovaries, and S2 cells were homogenized in NuPAGE® LDS sample
buffer (Invitrogen), heated for 5 minutes at 95°C and centrifuged. Samples were loaded on 10% NuPAGE®
(Invitrogen), and transferred to iBlot® nitrocellulose membranes (Invitrogen) following the manufacturer’s
instructions. Blocking and incubation of antibodies were done in 5% milk powder in PBT (Phosphate
Buffered Saline, pH 7.5, 0.05% Tween20). We used mouse anti-PIWI (1:10; Mikiko Siomi), mouse anti-
AUB (1:1000; Mikiko Siomi), mouse and rat anti- aTUB (1:500;Sigma and SEROTEC, respectively), and
rabbit anti-VASA (1:400; Ruth Lehmann). Secondary HRP-labeled IgG antibodies were obtained from
Jackson ImmunoResearch Laboratories. Antibody detection was done with the ECL Western blot detection
system (Amersham).

Immunohistochemical analysis

Immunofluerescence studies in larval brains were performed as described ( S27). In brief, third instar larval
brains were dissected in 0,7% NaCl, fixed for 30min in 4% PFA in 0,7% NaCl, and blocked in 10% fetal
calf serum in 0,7%NaCl, 0,1% Triton X-100 (blocking solution) for 2 hours at room temperature.
Antibodies were diluted in blocking solution. We used rabbit and rat anti-MIRA (1:1000), rabbit anti-
VASA (1:400; Ruth Lehmann), mouse anti-C(3)G (1:500; Scott Hawley), mouse anti-SQU(1:5000;
Hybridoma bank). Secondary Alexa Fluor 488 or Alexa Fluor 546 antibodies were obtained from
Invitrogen. DNA was stained with DAPI and the brains were mounted in Vectashield (Vector
Laboratories). We acquired images on a Leica SP2 confocal microscope and processed them with Adobe
Photoshop.

Protein quantification

Samples were prepared by homogenizing 1 larval brain in 10µl of sterilized H20. Protein concentration was
determined in a Nanodrop ND-1000 Spectrophotometer.

Allograft assay

Tissue dissection and injection was carried out as described ( S28). The glass needle had a diameter of
about 90 µm, and its tip was sharpened with a pair of tweezers. A pressure-injection system was made by
inserting the needle through a holder into a piece of silicon tubing that had a mouthpiece at the other end.
Donor third instar larvae were washed in distilled water, and their brains were dissected in 0,7% NaCl on a
siliconized microscope slide, and cut into small pieces. Young female adult hosts were anesthetized with
CO2 and stuck on a microscope slide, ventral side up, with double-sided sticky tape. A piece of larval brain
was picked up with the tip of the needle and injected tangentially in the mid-ventral part of the abdomen.
Implanted hosts were kept at 29°C.

Hierarchical clustering plot derived from genome-wide gene expression profiles of mbt tumors and control brains.

Three clusters can be seen, each containing control larval brains from w1118 larvae raised at 25°C, and l(3)mbtts1 and l(3)mbtE2 larvae raised at 17°C; mbt larval brain tumors from l(3)mbtts1, l(3)mbtE2 , and l(3)mbtts1 / l(3)mbtE2 larvae raised at 29°C; and l(3)mbtts1 tumors kept in allograft culture for one (T1), five (T5) and ten (T10) consecutive rounds.

Gene function and fold change range (FC minimum-maximum) of germline genes overexpressed in mbt tumors. Both mbt signature (MBTS) genes and non-MBTS genes are shown. Gene function is shown as described in FlyBase (http://www.FlyBase.org).

Work in our laboratory is supported by ONCASYM-037398, BFU2006-05813, BFU2009-07975, SGR2005, SRG200, CEN-20091016, and Consolider-Ingenio CSD2006-23. A.J. is a recipient of a Ministerio de Ciencia e Innovacion Formacion de Personal Investigador fellowship. Array data sets are deposited at Gene Expression Omnibus (accession no. GSE24917).

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:

"Germ Cell Genes and Cancer",  Science 24 December 2010: 1761-1762.

This fascinating study by Ana Janic, Leire Mendizabal, Salud Llamazares, David Rossell, and Cayetano Gonzalez reveals the active expression of germ cell and other embryonic genes withiu adult brain neoplasms in Drosophila species. The removal of such gene activity results in a decreased activity of the neoplasm. These phenomena characterize the formation and activity of embryomas, multi-gene networks of embryonic genes within adult neoplastic cells. It may be that all adult neoplasms require the activity of embryonic genes for their establishment and metastatic progression. RNA control molecules, specific for certain embryonic genes, are therapeutic for certain adult human neoplasms.

Additional References:

1. 1. Frenster JH, and Hovsepian JA,  ( 2007 )
    “Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms”.

2. Frenster JH, and Hovsepian JA,  ( 2008 )
    "Models of  Embryonic RNA Initiating and Reverting Adult Neoplasms".

3. Frenster JH, and Hovsepian JA,  ( 2008 )
     "Micro RNAs and adult neoplasms of embryonic type".

4. Hovsepian JA, and Frenster JH,  ( 2009 )
     "Genomic Models of Functional Embryomas within Adult Neoplastic Cells".

5. Frenster JH, and Hovsepian JA, ( 2009 )
     "Functional Embryomas as a Result of Embryonic Gene Re-Expression".

6. Frenster JH, and Hovsepian JA, ( 2010 )
     "Cellular Dynamics of Embryomas within Adult Neoplasms".

7. Frenster JH, and Hovsepian JA,  ( 2010 )
      "Analysis of Intra-Nuclear Entropy Changes during EMT Activation".

8. Hovsepian JA, and Frenster JH, ( 2010 )
      Heterochromatin -to- Euchromatin Transition ( H-ET ) during Gene Activation.

9. Frenster JH, and Hovsepian JA,  ( 2010 )
      "The Biophysics of the Cancer Cell".

10. Frenster JH, and Hovsepian JA,
       "Reprogramming and Neoplasia".

11. Ziosi M, Baena-López LA, Grifoni D, Froldi F, Pession A, Garoia F, Trotta V, Bellosta P, Cavicchi S, and Pession A, ( 2010 ),
"dMyc Functions Downstream of Yorkie to Promote the Supercompetitive Behavior of Hippo Pathway Mutant Cells".

12. Frenster JH, and Hovsepian JA, (2010 )
"Reprogramming the human cancer cell nucleus".

1. Each cell retains all of its embryonic genes for a lifetime.

2. Controls for embryonic genes are often absent in adults.

3. Uncontrolled embryonic genes can replicate wildly.

4.  Replicating genes participate in  intra-cellular competition.

5.  The basis for gene competition is selective transcription.

6.  MicroRNAs can reprogram embryomic transcription.

7.  Gene reprogramming can produce normal phenotypes.

8.  Normal phenotypes can by-pass chromosomal lesions.

9.  MicroRNA therapy may need to be permanent.

10. Transplantation of microRNAs could be preferred.

http://www.embryomas.net/

1. Pathways within cell genomes involve a flow of information.

2. Information can flow by direct contact or by third parties.

3. Direct contact within whole genomes is difficult to regulate.

4. DNA-DNA direct contects are influenced by agents.

5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.

6. Third parties within genomes involve RNAs and proteins.

7.  RNAs and proteins are easy to regulate or reverse.

8.  Information can be shared, lost, or transformed.

9. System information can be hidden during system isolation.

10.  Local information can be permanently lost during system entropy.

http://www.cancerbiophysics.net/

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