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Posts Tagged ‘Proceedings of the National Academy of Sciences of the United States of America’


Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Curator, Writer: Stephen J. Williams, Ph.D.

lung cancer

(photo credit: cancer.gov)

A report Lung Cancer Genome Surveys Find Many Potential Drug Targets, in the NCI Bulletin,

http://www.cancer.gov/ncicancerbulletin/091812/page2

summarizes the clinical importance of five new lung cancer genome sequencing projects. These studies have identified genetic and epigenetic alterations in hundreds of lung tumors, of which some alterations could be taken advantage of using currently approved medications.

The reports, all published this month, included genomic information on more than 400 lung tumors. In addition to confirming genetic alterations previously tied to lung cancer, the studies identified other changes that may play a role in the disease.

Collectively, the studies covered the main forms of the disease—lung adenocarcinomas, squamous cell cancers of the lung, and small cell lung cancers.

“All of these studies say that lung cancers are genomically complex and genomically diverse,” said Dr. Matthew Meyerson of Harvard Medical School and the Dana-Farber Cancer Institute, who co-led several of the studies, including a large-scale analysis of squamous cell lung cancer by The Cancer Genome Atlas (TCGA) Research Network.

Some genes, Dr. Meyerson noted, were inactivated through different mechanisms in different tumors. He cautioned that little is known about alterations in DNA sequences that do not encode genes, which is most of the human genome.

Four of the papers are summarized below, with the first described in detail, as the Nature paper used a multi-‘omics strategy to evaluate expression, mutation, and signaling pathway activation in a large cohort of lung tumors. A literature informatics analysis is given for one of the papers.  Please note that links on GENE names usually refer to the GeneCard entry.

Paper 1. Comprehensive genomic characterization of squamous cell lung cancers[1]

The Cancer Genome Atlas Research Network Project just reported, in the journal Nature, the results of their comprehensive profiling of 230 resected lung adenocarcinomas. The multi-center teams employed analyses of

  • microRNA
  • Whole Exome Sequencing including
    • Exome mutation analysis
    • Gene copy number
    • Splicing alteration
  • Methylation
  • Proteomic analysis

Summary:

Some very interesting overall findings came out of this analysis including:

  • High rates of somatic mutations including activating mutations in common oncogenes
  • Newly described loss of function MGA mutations
  • Sex differences in EGFR and RBM10 mutations
  • driver roles for NF1, MET, ERBB2 and RITI identified in certain tumors
  • differential mutational pattern based on smoking history
  • splicing alterations driven by somatic genomic changes
  • MAPK and PI3K pathway activation identified by proteomics not explained by mutational analysis = UNEXPLAINED MECHANISM of PATHWAY ACTIVATION

however, given the plethora of data, and in light of a similar study results recently released, there appears to be a great need for additional mining of this CGAP dataset. Therefore I attempted to curate some of the findings along with some other recent news relevant to the surprising findings with relation to biomarker analysis.

Makeup of tumor samples

230 lung adenocarcinomas specimens were categorized by:

Subtype

33% acinar

25% solid

14% micro-papillary

9% papillary

8% unclassified

5% lepidic

4% invasive mucinous
Gender

Smoking status

81% of patients reported past of present smoking

The authors note that TCGA samples were combined with previous data for analysis purpose.

A detailed description of Methodology and the location of deposited data are given at the following addresses:

Publication TCGA Web Page: https://tcga-data.nci.nih.gov/docs/publications/luad_2014/

Sequence files: https://cghub.ucsc.edu

Results:

Gender and Smoking Habits Show different mutational patterns

 

WES mutational analysis

  1. a) smoking status

– there was a strong correlations of cytosine to adenine nucleotide transversions with past or present smoking. In fact smoking history separated into transversion high (past and previous smokers) and transversion low (never smokers) groups, corroborating previous results.

mutations in groups              Transversion High                   Transversion Low

TP53, KRAS, STK11,                 EGFR, RB1, PI3CA

     KEAP1, SMARCA4 RBM10

 

  1. b) Gender

Although gender differences in mutational profiles have been reported, the study found minimal number of significantly mutated genes correlated with gender. Notably:

  • EGFR mutations enriched in female cohort
  • RBM10 loss of function mutations enriched in male cohort

Although the study did not analyze the gender differences with smoking patterns, it was noted that RBM10 mutations among males were more prevalent in the transversion high group.

Whole exome Sequencing and copy number analysis reveal Unique, Candidate Driver Genes

Whole exome sequencing revealed that 62% of tumors contained mutations (either point or indel) in known cancer driver genes such as:

KRAS, EGFR, BRMF, ERBB2

However, authors looked at the WES data from the oncogene-negative tumors and found unique mutations not seen in the tumors containing canonical oncogenic mutations.

Unique potential driver mutations were found in

TP53, KEAP1, NF1, and RIT1

The genomics and expression data were backed up by a proteomics analysis of three pathways:

  1. MAPK pathway
  2. mTOR
  3. PI3K pathway

…. showing significant activation of all three pathways HOWEVER the analysis suggested that activation of signaling pathways COULD NOT be deduced from DNA sequencing alone. Phospho-proteomic analysis was required to determine the full extent of pathway modification.

For example, many tumors lacked an obvious mutation which could explain mTOR or MAPK activation.

 

Altered cell signaling pathways included:

  • Increased MAPK signaling due to activating KRAS
  • Higher mTOR due to inactivating STK11 leading to increased proliferation, translation

Pathway analysis of mutations revealed alterations in multiple cellular pathways including:

  • Reduced oxidative stress response
  • Nucleosome remodeling
  • RNA splicing
  • Cell cycle progression
  • Histone methylation

Summary:

Authors noted some interesting conclusions including:

  1. MET and ERBB2 amplification and mutations in NF1 and RIT1 may be unique driver events in lung adenocarcinoma
  2. Possible new drug development could be targeted to the RTK/RAS/RAF pathway
  3. MYC pathway as another important target
  4. Cluster analysis using multimodal omics approach identifies tumors based on single-gene driver events while other tumor have multiple driver mutational events (TUMOR HETEROGENEITY)

Paper 2. A Genomics-Based Classification of Human Lung Tumors[2]

The paper can be found at

http://stm.sciencemag.org/content/5/209/209ra153

by The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM),*,

Paper Summary

This sequencing project revealed discrepancies between histologic and genomic classification of lung tumors.

Methodology

– mutational analysis by whole exome sequencing of 1255 lung tumors of histologically

defined subtypes

– immunohistochemistry performed to verify reclassification of subtypes based on sequencing data

Results

  • 55% of all cases had at least one oncogenic alteration amenable to current personalized treatment approaches
  • Marked differences existed between cluster analysis within and between preclassified histo-subtypes
  • Reassignment based on genomic data eliminated large cell carcinomas
  • Prospective classification of 5145 lung cancers allowed for genomic classification in 75% of patients
  • Identification of EGFR and ALK mutations led to improved outcomes

Conclusions:

It is feasible to successfully classify and diagnose lung tumors based on whole exome sequencing data.

Paper 3. Genomic Landscape of Non-Small Cell Lung Cancer in Smokers and Never-Smokers[3]

A link to the paper can be found here with Graphic Summary: http://www.cell.com/cell/abstract/S0092-8674%2812%2901022-7?cc=y?cc=y

Methodology

  • Whole genome sequencing and transcriptome sequencing of cancerous and adjacent normal tissues from 17 patients with NSCLC
  • Integrated RNASeq with WES for analysis of
    • Variant analysis
    • Clonality by variant allele frequency anlaysis
    • Fusion genes
  • Bioinformatic analysis

Results

  • 3,726 point mutations and more than 90 indels in the coding sequence
  • Smokers with lung cancer show 10× the number of point mutations than never-smokers
  • Novel lung cancer genes, including DACH1, CFTR, RELN, ABCB5, and HGF were identified
  • Tumor samples from males showed high frequency of MYCBP2 MYCBP2 involved in transcriptional regulation of MYC.
  • Variant allele frequency analysis revealed 10/17 tumors were at least biclonal while 7/17 tumors were monoclonal revealing majority of tumors displayed tumor heterogeneity
  • Novel pathway alterations in lung cancer include cell-cycle and JAK-STAT pathways
  • 14 fusion proteins found, including ROS1-ALK fusion. ROS1-ALK fusions have been frequently found in lung cancer and is indicative of poor prognosis[4].
  • Novel metabolic enzyme fusions
  • Alterations were identified in 54 genes for which targeted drugs are available.           Drug-gable mutant targets include: AURKC, BRAF, HGF, EGFR, ERBB4, FGFR1, MET, JAK2, JAK3, HDAC2, HDAC6, HDAC9, BIRC6, ITGB1, ITGB3, MMP2, PRKCB, PIK3CG, TERT, KRAS, MMP14

Table. Validated Gene-Fusions Obtained from Ref-Seq Data

Note: Gene columns contain links for GeneCard while Gene function links are to the    gene’s GO (Gene Ontology) function.

GeneA (5′) GeneB (3′) GeneA function (link to Gene Ontology) GeneB function (link to Gene Ontology) known function (refs)
GRIP1 TNIP1 glutamate receptor IP transcriptional repressor
SGMS1 STK10 sphingolipid synthesis ser/thr kinase
RASSF3 TTYH2 GTP-binding protein chloride anion channel
KDELR2 ROS1, GOPC ER retention seq. binding proto-oncogenic tyr kinase
ACSL4 DCAF6 fatty acid synthesis ?
MARCH8 PRKG1 ubiquitin ligase cGMP dependent protein kinase
APAF1 UNC13B, TLN1 caspase activation cytoskeletal
EML4 ALK microtubule protein tyrosine kinase
EDR3,PHC3 LOC441601 polycomb pr/DNA binding ?
DKFZp761L1918,RHPN2 ANKRD27 Rhophilin (GTP binding pr ankyrin like
VANGL1 HAO2 tetraspanin family oxidase
CACNA2D3 FLNB VOC Ca++ channel filamin (actin binding)

Author’s Note:

There has been a recent literature on the importance of the EML4-ALK fusion protein in lung cancer. EML4-ALK positive lung tumors were found to be les chemo sensitive to cytotoxic therapy[5] and these tumor cells may exhibit an epitope rendering these tumors amenable to immunotherapy[6]. In addition, inhibition of the PI3K pathway has sensitized EMl4-ALK fusion positive tumors to ALK-targeted therapy[7]. EML4-ALK fusion positive tumors show dependence on the HSP90 chaperone, suggesting this cohort of patients might benefit from the new HSP90 inhibitors recently being developed[8].

Table. Significantly mutated genes (point mutations, insertions/deletions) with associated function.

Gene Function
TP53 tumor suppressor
KRAS oncogene
ZFHX4 zinc finger DNA binding
DACH1 transcription factor
EGFR epidermal growth factor receptor
EPHA3 receptor tyrosine kinase
ENSG00000205044
RELN cell matrix protein
ABCB5 ABC Drug Transporter

Table. Literature Analysis of pathways containing significantly altered genes in NSCLC reveal putative targets and risk factors, linkage between other tumor types, and research areas for further investigation.

Note: Significantly mutated genes, obtained from WES, were subjected to pathway analysis (KEGG Pathway Analysis) in order to see which pathways contained signicantly altered gene networks. This pathway term was then used for PubMed literature search together with terms “lung cancer”, “gene”, and “NOT review” to determine frequency of literature coverage for each pathway in lung cancer. Links are to the PubMEd search results.

KEGG pathway Name # of PUBMed entries containing Pathway Name, Gene ANDLung Cancer
Cell cycle 1237
Cell adhesion molecules (CAMs) 372
Glioma 294
Melanoma 219
Colorectal cancer 207
Calcium signaling pathway 175
Prostate cancer 166
MAPK signaling pathway 162
Pancreatic cancer 88
Bladder cancer 74
Renal cell carcinoma 68
Focal adhesion 63
Regulation of actin cytoskeleton 34
Thyroid cancer 32
Salivary secretion 19
Jak-STAT signaling pathway 16
Natural killer cell mediated cytotoxicity 11
Gap junction 11
Endometrial cancer 11
Long-term depression 9
Axon guidance 8
Cytokine-cytokine receptor interaction 8
Chronic myeloid leukemia 7
ErbB signaling pathway 7
Arginine and proline metabolism 6
Maturity onset diabetes of the young 6
Neuroactive ligand-receptor interaction 4
Aldosterone-regulated sodium reabsorption 2
Systemic lupus erythematosus 2
Olfactory transduction 1
Huntington’s disease 1
Chemokine signaling pathway 1
Cardiac muscle contraction 1
Amyotrophic lateral sclerosis (ALS) 1

A few interesting genetic risk factors and possible additional targets for NSCLC were deduced from analysis of the above table of literature including HIF1-α, mIR-31, UBQLN1, ACE, mIR-193a, SRSF1. In addition, glioma, melanoma, colorectal, and prostate and lung cancer share many validated mutations, and possibly similar tumor driver mutations.

KEGGinliteroanalysislungcancer

 please click on graph for larger view

Paper 4. Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing[9]

For full paper and graphical summary please follow the link: http://www.cell.com/cell/abstract/S0092-8674%2812%2901061-6

Highlights

  • Exome and genome characterization of somatic alterations in 183 lung adenocarcinomas
  • 12 somatic mutations/megabase
  • U2AF1, RBM10, and ARID1A are among newly identified recurrently mutated genes
  • Structural variants include activating in-frame fusion of EGFR
  • Epigenetic and RNA deregulation proposed as a potential lung adenocarcinoma hallmark

Summary

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Paper 5. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer[10]

Highlights

  • Whole exome and transcriptome (RNASeq) sequencing 29 small-cell lung carcinomas
  • High mutation rate 7.4 protein-changing mutations/million base pairs
  • Inactivating mutations in TP53 and RB1
  • Functional mutations in CREBBP, EP300, MLL, PTEN, SLIT2, EPHA7, FGFR1 (determined by literature and database mining)
  • The mutational spectrum seen in human data also present in a Tp53-/- Rb1-/- mouse lung tumor model

 

Curator Graphical Summary of Interesting Findings From the Above Studies

DGRAPHICSUMMARYNSLCSEQPOST

The above figure (please click on figure) represents themes and findings resulting from the aforementioned studies including

questions which will be addressed in Future Posts on this site.

References:

  1. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012, 489(7417):519-525.
  2. A genomics-based classification of human lung tumors. Science translational medicine 2013, 5(209):209ra153.
  3. Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, Maher CA, Fulton R, Fulton L, Wallis J et al: Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012, 150(6):1121-1134.
  4. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, Asaka R, Hamanaka W, Ninomiya H, Uehara H et al: RET, ROS1 and ALK fusions in lung cancer. Nature medicine 2012, 18(3):378-381.
  5. Morodomi Y, Takenoyama M, Inamasu E, Toyozawa R, Kojo M, Toyokawa G, Shiraishi Y, Takenaka T, Hirai F, Yamaguchi M et al: Non-small cell lung cancer patients with EML4-ALK fusion gene are insensitive to cytotoxic chemotherapy. Anticancer research 2014, 34(7):3825-3830.
  6. Yoshimura M, Tada Y, Ofuzi K, Yamamoto M, Nakatsura T: Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene. Oncology reports 2014, 32(1):33-39.
  7. Yang L, Li G, Zhao L, Pan F, Qiang J, Han S: Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2014.
  8. Workman P, van Montfort R: EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence. Cancer discovery 2014, 4(6):642-645.
  9. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A et al: Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012, 150(6):1107-1120.
  10. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, Plenker D, Leenders F, Sun R, Zander T et al: Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics 2012, 44(10):1104-1110.

Other posts on this site which refer to Lung Cancer and Cancer Genome Sequencing include:

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

US Personalized Cancer Genome Sequencing Market Outlook 2018 –

Comprehensive Genomic Characterization of Squamous Cell Lung Cancers

International Cancer Genome Consortium Website has 71 Committed Cancer Genome Projects Ongoing

Non-small Cell Lung Cancer drugs – where does the Future lie?

Lung cancer breathalyzer trialed in the UK

Diagnosing Lung Cancer in Exhaled Breath using Gold Nanoparticles

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

Read Full Post »

Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com


Compilation of References by Leaders in Pharmaceutical Business Intelligence in the Journal http://pharmaceuticalintelligence.com about
Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation

Curator: Larry H Bernstein, MD, FCAP

Proteomics

  1. The Human Proteome Map Completed

Reporter and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/28/the-human-proteome-map-completed/

  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/06/24/proteomics-the-pathway-to-
understanding-and-decision-making-in-medicine/

3. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-         of-therapeutic-targets/

  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-                metabolome/

5. Genomics, Proteomics and standards

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/06/genomics-proteomics-and-standards/

6. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

 

Metabolomics

  1. Extracellular evaluation of intracellular flux in yeast cells

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2014/08/25/extracellular-evaluation-of-intracellular-flux-in-yeast-cells/

  1. Metabolomic analysis of two leukemia cell lines. I.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2014/08/23/metabolomic-analysis-of-two-leukemia-cell-lines-_i/

  1. Metabolomic analysis of two leukemia cell lines. II.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2014/08/24/metabolomic-analysis-of-two-leukemia-cell-lines-ii/

  1. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator, Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/22/metabolomics-metabonomics-and-functional-nutrition-the-next-step-          in-nutritional-metabolism-and-biotherapeutics/

  1. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Reviewer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/21/pentose-shunt-electron-transfer-galactose-more-lipids-in-brief/

  1. Mitochondria: More than just the “powerhouse of the cell”

Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

  1. Mitochondrial fission and fusion: potential therapeutic targets?

Ritu saxena

https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

4.  Mitochondrial mutation analysis might be “1-step” away

Ritu Saxena

https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

  1. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-                     leaders-in-pharmaceutical-intelligence/

  1. Metabolic drivers in aggressive brain tumors

Prabodh Kandal, PhD

https://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/

  1. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

Writer and Curator, Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-                        information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/

  1. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Larry H Bernstein, MD, FCAP, author and curator

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-            glycolysis-metabolic-adaptation/

  1. Therapeutic Targets for Diabetes and Related Metabolic Disorders

Reporter, Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/

10.  Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

11. The multi-step transfer of phosphate bond and hydrogen exchange energy

Larry H. Bernstein, MD, FCAP, Curator:

https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-bond-and-hydrogen-                          exchange-energy/

12. Studies of Respiration Lead to Acetyl CoA

https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

13. Lipid Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/15/lipid-metabolism/

14. Carbohydrate Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

15. Update on mitochondrial function, respiration, and associated disorders

Larry H. Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                   disorders/

16. Prologue to Cancer – e-book Volume One – Where are we in this journey?

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/13/prologue-to-cancer-ebook-4-where-are-we-in-this-journey/

17. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-          how-we-got-here/

18. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/11/01/inhibition-of-the-cardiomyocyte-specific-kinase-tnni3k/

19. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/05/15/the-binding-of-oligonucleotides-in-dna-and-3-d-lattice-structures/

20. Mitochondrial Metabolism and Cardiac Function

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

21. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/

22. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

Author and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-         tumor-growth-in-vivo/

23. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-                         conundrum/

24. Mitochondrial Damage and Repair under Oxidative Stress

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

25. Nitric Oxide and Immune Responses: Part 2

Author and Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

26. Overview of Posttranslational Modification (PTM)

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/

27. Malnutrition in India, high newborn death rate and stunting of children age under five years

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-                   children-age-under-five-years/

28. Update on mitochondrial function, respiration, and associated disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                  disorders/

29. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-         in-renal-disease/

30. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-                                  translational_medicine-part_2/

31. Epilogue: Envisioning New Insights in Cancer Translational Biology
Series C: e-Books on Cancer & Oncology

Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant

https://pharmaceuticalintelligence.com/2014/03/29/epilogue-envisioning-new-insights/

32. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone                         and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-                    hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy

33. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-                           related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
and Article Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-      and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-                    contractile/

34. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

35. Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-                           cytoskeleton/

36. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-              End-Stage/

37. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-               immunology/

38. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/ido-for-commitment-of-a-life-time-the-origins-and-mechanisms-of-             ido-indolamine-2-3-dioxygenase/

39. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP

https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-           of-immune-responses-for-good-and-bad/

40. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/06/26/signaling-pathway-that-makes-young-neurons-connect-was-                     discovered-scripps-research-institute/

41. Naked Mole Rats Cancer-Free

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/06/20/naked-mole-rats-cancer-free/

42. Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

https://pharmaceuticalintelligence.com/2013/05/06/alzheimers-disease-and-one-carbon-metabolism/

43. Problems of vegetarianism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

https://pharmaceuticalintelligence.com/2013/04/22/problems-of-vegetarianism/

44.  Amyloidosis with Cardiomyopathy

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/03/31/amyloidosis-with-cardiomyopathy/

45. Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/03/10/liver-endoplasmic-reticulum-stress-and-hepatosteatosis/

46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/

47. Nitric Oxide Function in Coagulation – Part II

Curator and Author: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/

48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/

49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/

50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/

51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

52. Mitochondrial Damage and Repair under Oxidative Stress

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

53. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-                 century-view/

54. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                  proteolysis-and-cell-apoptosis/

55. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis-reconsidered/

56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/

59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-         a-concomitant-influence-on-mitochondrial-function/

60. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy

Curator and Author: Ziv Raviv, PhD, RN 04/06/2013

https://pharmaceuticalintelligence.com/2013/04/06/targeting-mitochondrial-bound-hexokinase-for-cancer-therapy/

61. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/biochemistry-of-the-coagulation-cascade-and-platelet-aggregation/

Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/06/what-is-the-meaning-of-so-many-rnas/

  1. RNA and the transcription the genetic code

Larry H. Bernstein, MD, FCAP, Writer and Curator

https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

  1. A Primer on DNA and DNA Replication

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/29/a_primer_on_dna_and_dna_replication/

4. Synthesizing Synthetic Biology: PLOS Collections

Reporter: Aviva Lev-Ari

https://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

5. Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/

6. RNA and the transcription the genetic code

Writer and Curator, Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

7. A Great University engaged in Drug Discovery: University of Pittsburgh

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2014/07/15/a-great-university-engaged-in-drug-discovery/

8. microRNA called miRNA-142 involved in the process by which the immature cells in the bone  marrow give                              rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/24/microrna-called-mir-142-involved-in-the-process-by-which-the-                   immature-cells-in-the-bone-marrow-give-rise-to-all-the-types-of-blood-cells-including-immune-cells-and-the-oxygen-             bearing-red-blood-cells/

9. Genes, proteomes, and their interaction

Larry H. Bernstein, MD, FCAP, Writer and Curator

https://pharmaceuticalintelligence.com/2014/07/28/genes-proteomes-and-their-interaction/

10. Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator

https://pharmaceuticalintelligence.com/2014/07/29/regulation-of-somatic-stem-cell-function/

11. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/07/10/scientists-discover-that-pluripotency-factor-nanog-is-also-active-in-           adult-organisms/

12. Bzzz! Are fruitflies like us?

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/07/bzzz-are-fruitflies-like-us/

13. Long Non-coding RNAs Can Encode Proteins After All

Larry H Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/06/29/long-non-coding-rnas-can-encode-proteins-after-all/

14. Michael Snyder @Stanford University sequenced the lymphoblastoid transcriptomes and developed an
allele-specific full-length transcriptome

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/014/06/23/michael-snyder-stanford-university-sequenced-the-lymphoblastoid-            transcriptomes-and-developed-an-allele-specific-full-length-transcriptome/

15. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H                                     Bernstein, MD, FCAP

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/16/commentary-on-biomarkers-for-genetics-and-genomics-of-                        cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/

16. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies

Author an curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/05/18/observations-on-finding-the-genetic-links/

17. Silencing Cancers with Synthetic siRNAs

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/

18. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/12/cardiometabolic-syndrome-and-the-genetics-of-hypertension-the-neuroendocrine-transcriptome-control-points/

19. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-           mellitus-and-treatment-targets/

20. Loss of normal growth regulation

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/loss-of-normal-growth-regulation/

21. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/11/15/ct-angiography-truevision-metabolomics-genomic-phenotyping-for-           new-therapeutic-targets-to-atherosclerosis/

22.  CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

Genomics Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/30/cracking-the-code-of-human-life-the-birth-of-bioinformatics-                      computational-genomics/

23. Big Data in Genomic Medicine

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

24. From Genomics of Microorganisms to Translational Medicine

Author and Curator: Demet Sag, PhD

https://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-                      microorganisms-to-translational-medicine/

25. Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/01/06/summary-of-genomics-and-medicine-role-in-cardiovascular-diseases/

 26. Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious                      Depression

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/02/19/genomic-promise-for-neurodegenerative-diseases-dementias-autism-        spectrum-schizophrenia-and-serious-depression/

 27.  BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-         in-transcription-ubiquitination-and-dna-repair/

28. Personalized medicine gearing up to tackle cancer

Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/01/07/personalized-medicine-gearing-up-to-tackle-cancer/

29. Differentiation Therapy – Epigenetics Tackles Solid Tumors

Stephen J Williams, PhD

      https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

30. Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

     Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-          detection-treatment/

31. The Molecular pathology of Breast Cancer Progression

Tilde Barliya, PhD

https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression

32. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

33. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine –                                                       Part 1 (pharmaceuticalintelligence.com)

Aviva  Lev-Ari, PhD, RN

http://pharmaceuticalntelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

34. LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer                                         Personalized Treatment: Part 2

A Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-       drug-selection-in-cancer-personalized-treatment-part-2/

35. Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-        research-part-3/

36. Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of                           Cancer Scientific Leaders @http://pharmaceuticalintelligence.com

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/7000/Harnessing_Personalized_Medicine_for_ Cancer_Management-      Prospects_of_Prevention_and_Cure/

37.  GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico
effect of the inhibitor in its “virtual clinical trial”

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-             systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

38. Personalized medicine-based cure for cancer might not be far away

Ritu Saxena, PhD

  https://pharmaceuticalintelligence.com/2012/11/20/personalized-medicine-based-cure-for-cancer-might-not-be-far-away/

39. Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-         indexed-to-the-human-genome-sequence/

40. Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-                genomic-sequencing-to-cancer-diagnostics/

41. The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-         of-dna-wcrick-41953/

42. What can we expect of tumor therapeutic response?

Author and curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/12/05/what-can-we-expect-of-tumor-therapeutic-response/

43. Directions for genomics in personalized medicine

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

44. How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cancer-part1-transposon-            mediated-tumorigenesis/

45. mRNA interference with cancer expression

Author and Curator, Larry H. Bernstein, MD, FCAP

 https://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

46. Expanding the Genetic Alphabet and linking the genome to the metabolome

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-               metabolome/

47. Breast Cancer, drug resistance, and biopharmaceutical targets

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/18/breast-cancer-drug-resistance-and-biopharmaceutical-targets/

48.  Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression                            Analysis

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/breast-cancer-genomic-profiling-to-predict-survival-combination-of-           histopathology-and-gene-expression-analysis

49. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva  Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

50. Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-                   agricultural-biotechnology/

51. 2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2013_Genomics

52. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/Paradigm Shift in Human Genomics_/

Signaling Pathways

  1. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

  1. A Synthesis of the Beauty and Complexity of How We View Cancer:
    Cancer Volume One – Summary

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/03/26/a-synthesis-of-the-beauty-and-complexity-of-how-we-view-cancer/

  1. Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in
    serous endometrial tumors

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-ad-ubiquitin-           ligase-complex-genes-in-serous-endometrial-tumors/

4.  Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-              transition-in-prostate-cancer-cells/

5. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis/

6. Signaling and Signaling Pathways

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/

7.  Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/11/03/leptin-signaling-in-mediating-the-cardiac-hypertrophy-associated-            with-obesity/

  1. Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

  1. The Final Considerations of the Role of Platelets and Platelet Endothelial Reactions in Atherosclerosis and Novel
    Treatments

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/10/15/the-final-considerations-of-the-role-of-platelets-and-platelet-                      endothelial-reactions-in-atherosclerosis-and-novel-treatments

10.   Platelets in Translational Research – Part 1

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/10/07/platelets-in-translational-research-1/

11.  Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and
Cardiovascular Calcium Signaling Mechanism

Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to e-SERIES A:
Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-             smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

12. The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets

     Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to
e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and
Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-       kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-           differen/

13.  Nitric Oxide Signalling Pathways

Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/

14. Immune activation, immunity, antibacterial activity

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/immune-activation-immunity-antibacterial-activity/

15.  Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator

https://pharmaceuticalintelligence.com/2014/07/29/regulation-of-somatic-stem-cell-function/

16. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/07/10/scientists-discover-that-pluripotency-factor-nanog-is-also-active-in-adult-organisms/

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Heroes in Medical Research: Green Fluorescent Protein and the Rough Road in Science

Curator: Stephen J. Williams, Ph.D.

In this series, “Heroes in Medical Research”, I like to discuss the people who made some important contributions to science and medicine which underlie the great transformative changes but don’t usually get the notoriety given to Nobel Laureates or who seem to fly under the radar of popular news. Their work may be the development of research tools which allowed a great discovery leading to a line of transformative research, a moment of serendipity leading to discovery of a cure, or just contributions to the development of a new field or the mentoring of a new generation of scientists and clinicians. One such discovery, which has probably been pivotal in many of our research, is the discovery of the green fluorescent protein (GFP), commonly used as an invaluable tool to monitor protein for cellular expression and localization studies. Although the development of research tools, whether imaging tools, vectors, animal models, cell lines, and such are not heralded, they always assist in the pivotal discoveries of our time. The following is a heartwarming story by Discover Magazine’s Yudhijit Bhattacharjee behind Dr. Douglas Prasher’s discovery of the green fluorescent protein, his successful efforts to sequence the gene and subsequent struggles in science and finally scientific recognition for his work. In addition the story describes Dr. Prather’s perseverance, a trait necessary for every scientist.

http://discovermagazine.com/2011/apr/30-how-bad-luck-networking-cost-prasher-nobel

 

The following is a wonderful entry into Wikipedia about Dr. Prasher at:

http://en.wikipedia.org/wiki/Douglas_Prasher

including a listing of his publications including the seminal Science and PNAS publications1,2.

 

prasher

 

 

(Photo: Dr. Prasher in the lab at UCSD. Photo credit UCSD and John Galstaldo)

 

 

 

In summary, Dr. Prather had been working at Wood’s Hole in Massachusetts trying to discover, isolate, then clone the protein which allowed a species of jellyfish living in the cold waters of the North Pacific, Aequorea victoria, to emit a green glow. Eventually he cloned the GFP gene, but gave up on work to express the gene in mammalian cells. Before leaving Wood’s Hole he gave the gene to Dr. Roger Tsien, who with Dr. Martin Chalfie and Osamu Shimomura showed the utility of GFP as an intracellular tracer to visualize, in real time, the expression and localization of GFP-tagged proteins (all three shared the 2008 Nobel Prize for this work). Dr. Tsien however realized the importance of Douglas’s cloning work as pivotal for their research, contacted Douglas (who now due to the bad economy was working at a Toyota dealership in Alabama) and invited him to the Nobel Prize Award Ceremony in Sweden as his guest. Although Dr. Prasher had “left academic science” he never really stopped his quest for a scientific career, using his spare time to review manuscripts.

Other researchers have invited their colleagues who made important contributions to the ultimate Nobel work. One such guest was one of my colleagues Dr. Leonard Cohen, who worked with Dr. Irwin Rose and Avram Hershko at the Institute for Cancer Research in Philadelphia a cell-free system from clams to discover the mechanism how cyclin B is degraded during the exit from the cell cycle (from A. Hershko’s Nobel speech). Dr. Hershko had acknowledged a slew of colleagues and highlighted their contributions to the ultimate work. It shows how even small discoveries can contribute to the sphere of scientific knowledge and breakthrough.

Luckily, in the end, perseverance has paid off as Dr. Prasher is now using his talents in Roger Tsien‘s group at the University of California in San Diego.

References:

1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., Green fluorescent protein as a marker for gene expression. Science, 263(5148), 802-805 (1994).

 

2. Heim, R., Prasher, D.C., Tsien, R.Y., Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA, 91(26), 12501-12504 (1994).

More posts on this site on Heroes in Medical Research series include:

Heroes in Medical Research: Developing Models for Cancer Research

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

 

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The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

 

Updated 6/25/2019

Writer. Reporter: Stephen J. Williams, Ph.D.

gottingen minipig2

 

 

The choice of suitable animal model of disease may define future success or failure for drug development, basic and translational research, or biomarker discovery projects.   Indeed, as highlighted in one of my earlier posts “Heroes in Medical Research: Developing Models for Cancer Research”, the choice of animal to model a human disease can have drastic implications in the basic researchers ability to understand metabolic and genetic factors causally associated with disease development. As described in that post the King rat model led to our understanding of the genetics of early development and sex determination while early mouse models helped us to understand the impact of microenvironment on cell fate and the discovery of stem cells. In addition, transgenic and immunodeficient mice resulted in transformational studies on our understanding of cancer. Small rodent models are ideal for following reasons:

  • Ease of genetic manipulation
  • Availability of well-defined models
  • Ease of low cost of use

Regardless of these benefits many investigators in industry and academia are looking to models of human disease in animals more closely resembling human anatomy, physiology, and genetics.

 

There is a growing need for alternative animal models in cancer research.

 

As I had discussed in another of my earlier posts “The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research”, the pig is gaining notoriety and acceptance as a very suitable animal to model human disease as minipigs and humans have:

  • Similar physiology
  • Similar genetics: >90% homology
  • Similar anatomic dimensions: i.e. Adult Gottingen minipigs are 70kg (adult human male weight)
  • Similar organ size and structure to humans organ size and structure
  • Pig genome sequencing project nearly complete
  • Ability to manipulate pig genetics

The post had discussed the development of a severe combined immunodeficient (SCID) pig by investigators at Iowa State and Kansas State University. This line of pigs, selected on a specific diet, could act as recipients for human cancer cell lines, a proof of their SCID phenotype.

A report featured on Fierce Biotech Research “MU Scientists Successfully Transplant, Grow Stem Cells in Pigs” discussed the development of a new genetically-modified immunodeficient porcine model by researchers at the University of Missouri, recently published in Proceedings of the National Academy of Sciences[1].

These pigs are available from the National Swine Resource and Research Center (http://nsrrc.missouri.edu).

For the report on Fierce Biotech Research please follow the link below:

http://www.fiercebiotechresearch.com/press-releases/mu-scientists-successfully-transplant-grow-stem-cells-pigs

 

The report in FierceBiotech highlights the type of studies an immunocompromised pig model would be useful for including:

  • Regenerative medicine
  • Xenotransplantation
  • Tumor growth and efficacy studies

 

Comments in the post from the investigators explained the benefits of developing such a porcine model system including:

“The rejection of transplants and grafts by host bodies is a huge hurdle for medical researchers,” said R. Michael Roberts, Curators Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. “By establishing that these pigs will support transplants without the fear of rejection, we can move stem cell therapy research forward at a quicker pace.”

The studies main investigators, Drs. Randall Prather and R. Michael Roberts, both of University of Missouri, along with first authors Kiho Lee, Deug-Nam Kwon and Toshihiko Ezashi, used biallellic mutation of the RAG2 gene in Gottingen minipig fibroblasts and then subsequent somatic cell nuclear transfer (SCNT) to produce the RAG2-/- animals. (Rag2 is a protein involved in V(D)J recombination of antibodies during early B and T cell development. See GeneCard link above)

As proof of their SCID phenotype the authors showed that

  1. these RAG2-/- animals could act as host for human induced pluripotent stem cells
  2. act as recipient for allogeneic porcine stem cells
  3. reduced levels of (CD21+) B cells and (CD3+) T cells
  4. growth retardation if housed under standard, non-sterile conditions

Details of the study are given below:

Methodology Used

For Production of Gottingen minipigs carrying the RAG2 mutation

To produce targeted mutations in RAG2:

  • TALENS () were constructed to produced mutation in exon 2 of RAG2
  • Constructed TALENS and reporter electroporated in fetal-derived pig fibroblasts
  • SCNT used to transfer RAG2 mutant nuclei to donor oocytes
  • 9 embryo transfers resulted in 22 live piglets
  • Piglets genotyped as either monoallelic or biallelic RAG2 mutant
  • RAG2wild-type and mutants housed in either pathogen-free or normal housing conditions

To verify SCID phenotype of litter by either

  1. Graft acceptance of human iPSCs and teratoma formation

–          Fibroblasts from human umbilical cord reprogrammed to pluripotency; verified by pluripotent markers POUSF1, NANOG, SSEA-3)

–          Two human and porcine iPSC lines with trophoblastic properties[2] were injected subcutaneously in ear or flank

–          Tumor formation analyzed by immunohistochemistry using markers:

CTNNBI (B-catenin)

VWF (von Willebrand

DES and ACTG2

GFAP and ENO2

Human specific MFN1 (both antibody and gene primers)

  1. Flow Cytometry

–          Analysis of piglet spleen cells for B cell population (CD21)

–          Analysis of piglet spleen cell for T cell population (CD3)

C.    Histology

– histo evaluation of thymus, spleen

– marker evaluation of spleen using anti-CD79A (B cells), CD3 (T cells),

CD335 (NK cells)

Results

TALEN produced a variety of indels (insertion/deletions) and three RAG2 mutatnt colonies (containing monoallelic, mix of mono and biallelic) used for SCNT.

Three litters produced 16 piglets (eight survived [four mono and four biallelic]

Biallelic RAG2 mutants showed slower weight gain than wild type or monoallelic mutants with signs of inflammation and apoptosis in spleen and designated “failure to thrive” in standard housing…needed a clean environment to thrive.

Biallelic mutant pigs lacked mature CD21 B cells and CD3 T cells but contained macrophages and NK cells.

Implantation of human and allogenic porcine pluripotent stem cells (trophoblastic) showed rapid development of teratomas.
References

  1. Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, Brown AN, Samuel MS, Park KW, Walters EM et al: Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proceedings of the National Academy of Sciences of the United States of America 2014, 111(20):7260-7265.
  2. Ezashi T, Matsuyama H, Telugu BP, Roberts RM: Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells. Biology of reproduction 2011, 85(4):779-787.

Updated 6/25/2019

The following articles represent an update on the ability to create genetically predisposed porcine models of cancer.  The ability to utilize transposable elements to introduce genetic changes in porcine cells in combination with Somatic Cell Nuclear Transfer technology with the ultimate goal to create a transgenic minipig is discussed.  The next two articles describe the ability of the scid pig to act as a recipient for human ovarian cancer cells and description of a transgenic inducible porcine intestinal tumor model.

Transgenic Res. 2011 Jun;20(3):533-45. doi: 10.1007/s11248-010-9438-x. Epub 2010 Aug 29.

Pig transgenesis by Sleeping Beauty DNA transposition.

Jakobsen JE1Li JKragh PMMoldt BLin LLiu YSchmidt MWinther KDSchyth BDHolm IEVajta GBolund LCallesen HJørgensen ALNielsen ALMikkelsen JG.

Author information

Abstract

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

This paper makes use of two technologies: transposon mediated gene transfer to introduce foreign DNA, for example a disease predisposition gene, into oocytes or early embryos, without the use of viral vectors; and use of SCNT to clone a minipig from viable embryos.

 

The transposon mediated system is based on the Sleeping Beauty (SB) vector system, which is a cut and paste DNA transposon belonging to the Tc1/mariner superfamily of transposable elements(1).  Transposable DNA elements are mobile genetic elements which integrate into genomic DNA, in the case of the SB system into discrete sequence elements of actively transcribed genes.  The system consists of two entities: 1) a transposase responsible for cutting and pasting the mobile element and 2) the transposon; the vectorial DNA sequence which is inserted into genomic DNA.  SB transposition has been used to integrate exogenous genetic elements into the genome of various mammalian species(2) and to make tumor models in mice (3-7) and to transform, ex-vivo, porcine ovarian epithelial cells (8) and to stably integrate GFP containing vectors into porcine fibroblast genome(9).  Because of the efficiency and nonviral integration of exogenous vectors into mammalian systems, Sleeping Beauty system has been considered as a potential therapeutic gene transfer modality (10-12).

 

  1. Li, Z.H., Liu, D.P., Wang, J., Guo, Z.C., Yin, W.X., and Liang, C.C. Inversion and transposition of Tc1 transposon of C. elegans in mammalian cells. Somat Cell Mol Genet. 1998; 24:363-369.
  2. Balciuniene, J., Nagelberg, D., Walsh, K.T., Camerota, D., Georlette, D., Biemar, F., et al. Efficient disruption of Zebrafish genes using a Gal4-containing gene trap. BMC Genomics. 2013; 14:619.
  3. Romano, G., Marino, I.R., Pentimalli, F., Adamo, V., and Giordano, A. Insertional mutagenesis and development of malignancies induced by integrating gene delivery systems: implications for the design of safer gene-based interventions in patients. Drug News Perspect. 2009; 22:185-196.
  4. Dupuy, A.J. Transposon-based screens for cancer gene discovery in mouse models. Semin Cancer Biol. 2010; 20:261-268.
  5. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G., and Jenkins, N.A. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature. 2005; 436:221-226.
  6. Dupuy, A.J., Clark, K., Carlson, C.M., Fritz, S., Davidson, A.E., Markley, K.M., et al. Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci U S A. 2002; 99:4495-4499.
  7. Dupuy, A.J., Fritz, S., and Largaespada, D.A. Transposition and gene disruption in the male germline of the mouse. Genesis. 2001; 30:82-88.
  8. Hamilton, T.C., Williams, S.J., and Cvetkovic, D. 2010. Cancer Compositions, Animal Models, and Methods of Use Thereof. U.S.P. Office, editor. USA: Fox Chase Cancer Center.
  9. Clark, K.J., Carlson, D.F., Foster, L.K., Kong, B.W., Foster, D.N., and Fahrenkrug, S.C. Enzymatic engineering of the porcine genome with transposons and recombinases. BMC Biotechnol. 2007; 7:42.
  10. Ivics, Z., and Izsvak, Z. Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies. Methods Mol Biol. 2004; 260:255-276.
  11. Liu, H., Liu, L., Fletcher, B.S., and Visner, G.A. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. FASEB J. 2006; 20:2384-2386.
  12. Ohlfest, J.R., Lobitz, P.D., Perkinson, S.G., and Largaespada, D.A. Integration and long-term expression in xenografted human glioblastoma cells using a plasmid-based transposon system. Mol Ther. 2004; 10:260-268.

 

A second paper, by Larry Shook and Geoffrey Clark’s groups describe the production of ex vivo transformed porcine breast cancer line, driven by inactivation of BRCA1.  In this paper normal porcine breast epithelial cells were immortalized by transfection with SV large T antigen (SV-LT) and upon inactivation of porcine BRCA1 in these immortalized cell lines, developed phenotype characteristic of transformed cells and exhibited cancer stem cell characteristics.  The end point assay for transformation was growth in soft agar however the authors did not confirm malignancy in either SCID mice or SCID pigs.

Front Genet. 2015 Aug 25;6:269. doi: 10.3389/fgene.2015.00269. eCollection 2015.

A porcine model system of BRCA1 driven breast cancer.

Donninger H1Hobbing K2Schmidt ML3Walters E4Rund L5Schook L5Clark GJ2.

Author information

Abstract

BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1expression is down-regulated in ~30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer. In contrast to mice, pig biological systems, and cancer genetics appear to more closely resemble their human counterparts. To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.

 

 

Figure 1. Immortalization of pig mammary epithelial cells. Primary pig breast epithelial cells were stably transfected with an SV40 LT expression construct and selected in puromycin. Surviving cells were serially passaged to confirm immortalization.

 

fgene-06-00269-g001 immortalized breast porcine epithelial cells

 

 

Figure 3. Loss of BRCA1 enhances pig mammary epithelial cell growth. (A) Serially passaging the pig mammary epithelial cells stably knocked down for BRCA1 resulted in an altered morphology compared to those cells stably expressing the LacZ miRNA. (B) 2 × 104 cells/well were plated in 6-well plates and cell growth was determined by counting the number of cells at the indicated times. Error bars show standard error, p < 0.05.

fgene-06-00269-g003growthofbrcaminusporbrepith

 

 

Figure 5. Loss of BRCA1 enhances the transformed phenotype of pig mammary epithelial cells. (A) The pig breast epithelial cells stably expressing BRCA1 miRNA were plated in soft agar and scored for growth 14 days later. Representative photomicrographs are shown in the top panel and data from three independent experiments quantitated in the bar graph in the lower panel. (B) 1 × 106 cells/well were plated in polyHEMA-coated 12-well plates and cell viability assessed 48 h later by trypan blue staining. Error bars show standard error, p < 0.05.

fgene-06-00269-g005brca1minuporbrepithcolonies

 

A third paper describes the development, in Gottingen minipigs, of a transgenic inducible model of intestinal cancer.

Mol Oncol. 2017 Nov;11(11):1616-1629. doi: 10.1002/1878-0261.12136. Epub 2017 Oct 10.

A genetically inducible porcine model of intestinal cancer.

Callesen MM1Árnadóttir SS1Lyskjaer I1Ørntoft MW1Høyer S2Dagnaes-Hansen F3Liu Y4Li R4Callesen H4Rasmussen MH1Berthelsen MF3Thomsen MK3Schweiger PJ5Jensen KB5Laurberg S6Ørntoft TF1Elverløv-Jakobsen JE3Andersen CL1.

Author information

Abstract

Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT – which inhibits p53 – and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.

 

Other posts on this site related to Cancer Research Tools include

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

Heroes in Medical Research: Developing Models for Cancer Research

Reprogramming Induced Pleuripotent Stem Cells

The Cancer Research Concentration @ Leaders in Pharmaceutical Business Intelligence

A Synthesis of the Beauty and Complexity of How We View Cancer

Guidelines for the welfare and use of animals in cancer research

Gene Therapy and the Genetic Study of Disease: @Berkeley and @UCSF – New DNA-editing technology spawns bold UC initiative as Crispr Goes Global

 

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Heroes in Medical Research: Developing Models for Cancer Research

Author, Curator: Stephen J. Williams, Ph.D.

 

The current rapid progress in cancer research would have never come about if not for the dedication of past researchers who had developed many of the scientific tools we use today. In this issue of Heroes in Medical Research I would like to give tribute to the researchers who had developed the some of the in-vivo and in-vitro models which are critical for cancer research.

 

The Animal Modelers in Cancer Research

Helen Dean King, Ph.D. (1869-1955)

Helen Dean King

Helen Dean King, Ph.D. from www.ExplorePAhistory.com; photo Courtesy of the Wistar Institute Archive Collection, Philadelphia, PA

 

 

The work of Dr. Helen Dean King on rat inbreeding led to development of strains of laboratory animals. Dr. King taught at Bryn Mawr College, then worked at University of Pennsylvania and the Wistar Institute under famed geneticist Thomas Hunt Morgan, researching if inbreeding would produce harmful genetic traits.   At University of Pennsylvania she examined environmental and genetic factors on gender determination.

 

 

 

 

Important papers include [1-6]as well as the following contributions:

“Studies in Inbreeding”, “Life Processes in Gray Norway Rats During Fourteen Years in Captivity”, doctoral thesis on embryologic development in toads (1899)

 

Milestones include:

 

1909    started albino rat breeding and bred 20 female and male from same litter (King colony) to 25

successive generations (inbreeding did not cause harmful traits)

 

1919     started to domesticate the wild Norwegian rats that ran thru Philadelphia (six pairs Norway rats

thru 28 generations)

A good reference for definitions of rat inbreeding versus line generation including a history of Dr. King’s work can be found at the site: Munificent Mischief Rattery and a brief history here.[7] In addition, Dr. King had investigated using rat strains as a possible recipient for tumor cells. The work was an important advent to the use of immunodeficient models for cancer research.

 

As shown below Philadelphia became a hotbed for research into embryology, development, genetics, and animal model development.

 

Beatrice Mintz, Ph.D.

(Beatrice Minz, Ph.D.; photo credit Fox Chase Cancer Center, www.pubweb.fccc.edu) Mintz

Dr. Mintz, an embryologist and cancer researcher from Fox Chase Cancer Center in Philadelphia, PA, contributed some of the most seminal discoveries leading to our current understanding of genetics, embryo development, cellular differentiation, and oncogenesis, especially melanoma, while pioneering techniques which allowed the development of genetically modified mice.

If you get the privilege of hearing her talk, take advantage of it. Dr. Mintz is one of those brilliant scientists who have the ability to look at a clinical problem from the viewpoint of a basic biological question and, at the same time, has the ability to approach the well-thought out questions with equally well thought out experimental design. For example, Dr. Mintz asked if a cell’s developmental fate was affected by location in the embryo. This led to her work by showing teratocarcinoma tumor cells in the developing embryo could revert to a more normal phenotype, essentially proving two important concepts in development and tumor biology:

  1. The existence of pluripotent stem cells
  2. That tumor cells are affected by their environment (which led to future concepts of the importance of tumor microenvironment on tumor growth

Other seminal discoveries included:

  • Development of the first mouse chimeras using novel cell fusion techniques
  • With Rudolf Jaenisch in 1974, showed integration of viral DNA from SV40, could be integrated into the DNA of developing mice and persist into adulthood somatic cells, the first transgenesis in mice which led ultimately to:
  • Development of the first genetically modified mouse model of human melanoma in 1993

Her current work, seen on the faculty webpage here, is developing mice with predisposition to melanoma to uncover risk factors associated with the early development of melanoma.

In keeping with the Philadelphia tradition another major mouse model which became seminal to cancer drug discovery was co-developed in the same city, same institute and described in the next section.

It is interesting to note that the first cloning of an animal, a frog, had taken place at the Institute for Cancer Research, later becoming Fox Chase Cancer Center, which was performed by Drs. Robert Briggs and Thomas J. King and reported in the 152 PNAS paper Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs.[8]

 

 The Immunodeficient Animal as a Model System for Cancer Research – Dr. Mel Bosma, Ph.D.

 

Bosma

Melvin J. Bosma, Ph.D.; photo credit Fox Chase Cancer Center

In the summer of 1980 at Fox Chase Cancer Center, Dr. Melvin J. Bosma and his co-researcher wife Gayle discovered mice with deficiencies in common circulating antibodies and since, these mice were littermates, realized they had found a genetic defect which rendered the mice immunodeficient (upon further investigation these mice were unable to produce mature B and T cells). These mice were the first scid (severe combined immunodeficiency) colony. The scid phenotype was later found to be a result of a spontaneous mutation in the enzyme Prkdc {protein kinase, DNA activated, catalytic polypeptide} involved in DNA repair, and ultimately led to a defect in V(D)J recombination of immunoglobulins.

The emergence of this scid mouse was not only crucial for AIDS research but was another turning point in cancer research , as researchers now had a robust in-vivo recipient for human tumor cells. The orthotopic xenograft of human tumor cells now allowed for studies on genetic and microenvironmental factors affecting tumorigenicity, as well as providing a model for chemotherapeutic drug development (see Suggitt for review and references)[9]. A discussion of the pros and cons of the xenograft system for cancer drug discovery would be too voluminous for this post and would warrant a full review by itself. But before the advent of such scid mouse systems researchers relied on spontaneous and syngeneic mouse tumor models such as the B16 mouse melanoma and Lewis lung tumor model.

Other scid systems have been developed such as in the dog, horse, and pig. Please see the following post on this site The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research. The athymic (nude) mouse (nu/nu) also is a popular immunodeficient mouse model used for cancer research

Two other in-vivo tumor models: Patient Derived Xenografts (PDX) and Genetically Engineered Mouse models (GEM) deserve their own separate discussion however the success of these new models can be attributed to the hard work of the aforementioned investigators. Therefore I will post separately and curate PDX and GEM models of cancer and highlight some new models which are having great impact on cancer drug development.

 

References

1.         Loeb L, King HD: Transplantation and Individuality Differential in Strains of Inbred Rats. The American journal of pathology 1927, 3(2):143-167.

2.         Lewis MR, Aptekman PM, King HD: Retarding action of adrenal gland on growth of sarcoma grafts in rats. J Immunol 1949, 61(4):315-319.

3.         Aptekman PM, Lewis MR, King HD: Tumor-immunity induced in rats by subcutaneous injection of tumor extract. J Immunol 1949, 63(4):435-440.

4.         Lewis MR, Aptekman PM, King HD: Inactivation of malignant tissue in tumor-immune rats. J Immunol 1949, 61(4):321-326.

5.         Lewis MR, King HD, et al.: Further studies on oncolysis and tumor immunity in rats. J Immunol 1948, 60(4):517-528.

6.         Aptekman PM, Lewis MR, King HD: A method of producing in inbred albino rats a high percentage of immunity from tumors native in their strain. J Immunol 1946, 52:77-86.

7.         Ogilvie MB: Inbreeding, eugenics, and Helen Dean King (1869-1955). Journal of the history of biology 2007, 40(3):467-507.

8.         Briggs R, King TJ: Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs. Proceedings of the National Academy of Sciences of the United States of America 1952, 38(5):455-463.

9.         Suggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clinical cancer research : an official journal of the American Association for Cancer Research 2005, 11(3):971-981.

 

Other posts on this site about Cancer, Animal Models of Disease, and other articles in this series include:

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

A Synthesis of the Beauty and Complexity of How We View Cancer

Guidelines for the welfare and use of animals in cancer research

Importance of Funding Replication Studies: NIH on Credibility of Basic Biomedical Studies

FDA Guidelines For Developmental and Reproductive Toxicology (DART) Studies for Small Molecules

Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations:

What`s new in pancreatic cancer research and treatment?

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

Reuben Shaw, Ph.D., a geneticist and researcher at the Salk Institute: Metabolism Influences Cancer

 

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Loss of Gene Islands May Promote a Cancer Cell’s Survival, Proliferation and Evolution: A new Hypothesis (and second paper validating model) on Oncogenesis from the Elledge Laboratory

Writer, Curator: Stephen J. Williams, Ph.D.

It is well established that a critical event in the transformation of a cell to the malignant state involves the mutation of hosts of oncogenes and tumor suppressor genes, which in turn, confer on a cell the inability to properly control its proliferation.    On a genomic scale, these mutations can result in gene amplifications, loss of heterozygosity (LOH), and epigenetic changes resulting in tumorigenesis.  The “two hit hypothesis”, proposed by Dr. Al Knudson of Fox Chase Cancer Center[1], proposes that two mutations in the same gene are required for tumorigenesis, initially proposed to explain the progression of retinoblastoma in children, indicating a recessive disease.

(Excerpts from a great article explaining the two-hit-hypothesis is given at the end of this post).

And, although many tumor genomes display haploinsufficeint tumor suppressor genes, and fit the two hit model quite nicely, recent data show that most tumors display hemizygous recurrent deletions within their genomes.  Tumors display numerous recurrent hemizygous focal deletions that seem to contain no known tumor suppressor genes. For instance a recent analysis of over three thousand tumors including breast, bladder, pancreatic, ovarian and gastric cancers averaged greater than 10 deletions/tumor and 82 regions of recurrent focal deletions,

It has been proposed these great number of hemizygous deletions may be a result of:

  • a recessive tumor suppressor gene requiring mutation or silencing of second allele
  • the mutation may recur as they are located in fragile sites (unstable genomic regions)
  • single-copy loss may provide selective advantage regardless of the other allele

Note: some definitions of hemizygosity are given below.  In general at any locus, each parental chromosome can have 3 deletion states:

  1. wild type
  2. large deletion
  3. small deletion

Hemizygous deletions only involve one allele, not both alleles which is unlike the classic tumor suppressor like TP53

To see if it is possible that only one mutated allele of a tumor suppressor gene may be a casual event for tumorigenesis, Dr. Nicole Solimini and colleagues, from Dr. Stephen Elledge’s lab at Harvard, proposed a hypothesis they termed the cancer gene island model, after analyzing the regions of these hemizygous deletions for cancer related genes[2].  Dr. Soliin and colleagues analyzed whole-genome sequence data for 526 tumors in the COSMIC database comparing to a list generated from the Cancer Gene Census for homozygous loss-of-function mutations (mutations which result in a termination codon or frame-shift mutation: {this produces a premature stop in the protein or an altered sequence leading to a nonfunctional protein}.

Results of this analysis revealed:

  1. although tumors have a wide range of deletions per tumor (most epithelial high grade like ovarian, bladder, pancreatic, and esophageal adenocarcinomas had 10-14 deletions per tumor
  2. and although tumors exhibited a wide range (2- 16 ) loss of function mutations
  3. ONLY 14 of 82 recurrent deletions contained a known tumor suppressor gene and was a low frequency event
  4. Most recurrent cancer deletions do not contain putative tumor suppressor genes.

Therefore, as the authors suggest, an alternate method to the two-hit hypothesis may account for a selective growth advantage for these types of deletions, defining these low frequency hemizygous mutations in two general classes

  1. STOP genes: suppressors of tumor growth and proliferation
  2. GO genes: growth enhancers and oncogenes

Identifying potential STOP genes

To identify the STOP and GO genes the authors performed a primary screen of an shRNA library in telomerase (hTERT) immortalized human mammary epithelial cells using increased PROLIFERATION as a screening endpoint to determine STOP genes and decreased proliferation and lethality (essential genes) to determine possible GO genes. An initial screen identified 3582 possible STOP genes.  Using further screens and higher stringency criteria which focused on:

  • Only genes which increased proliferation in independent triplicate screens
  • Validated by competition assays
  • Were enriched more than four fold in three independent shRNA screens

the authors were able to focus on and validate 878 genes to determine the molecular pathways involved in proliferation.

These genes were involved in cell cycle regulation, apoptosis, and autophagy (which will be discussed in further posts).

To further validate that these putative STOP genes are relevant in human cancer, the list of validated STOP genes found in the screen was compared to the list of loss-of-function mutations in the 526 tumors in the COSMIC databaseSurprisingly, the validated STOP gene list were significantly enriched for known and possibly NOVEL tumor suppressor genes and especially loss of function and deletion mutations but also clustered in gene deletions in cancer.  This not only validated the authors’ model system and method but suggests that hemizygous deletions in multiple STOP genes may contribute to tumorigenesis

as the function of the majority of STOP genes is to restrain tumorigenesis

A few key conclusions from this study offer strength to an alternative view of oncogenesis NAMELY:

  • Loss of multiple STOP genes per deletion optimize a cancer cell’s proliferative capacity
  • Cancer cells display an insignificant loss of GO genes, minimizing negative impacts on cellular fitness
  • Haploinsufficiency in multiple STOP genes can result in similar alteration of function similar to complete loss of both alleles of
  • Cancer evolution may result from selection of hemizygous loss of high number of STOP and low number of GO genes
  • Leads to a CANCER GENE ISLAND model where there is a clonal evolution of transformed cells due to selective pressures

A link to the supplemental data containing STOP and GO genes found in validation screens and KEGG analysis can be found at the following link:

http://www.sciencemag.org/content/337/6090/104/suppl/DC1#

A link to an interview with the authors, originally posted on Harvard’s site can be found here.

Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns and Shape the Cancer Genome; a new paper from the Elledge group in the journal Cell

http://www.cell.com/retrieve/pii/S0092867413012877

A concern of the authors was the extent to which gene silencing could have on their model in tumors.  The validation of the model was performed in cancer cell lines and compared to tumor genome sequence in publicly available databases however a followup paper by the same group shows that haploinsufficiency contributes a greater impact on the cancer genome than these studies have suggested.

In a follow-up paper by the Elledge group in the journal Cell[3], Theresa Davoli and colleagues, after analyzing 8,200 tumor-normal pairs, show there are many more cancer driver genes than once had been predicted.  In addition, the distribution and potency of STOP genes, oncogenes, and essential genes (GO) contribute to the complex picture of aneuploidy seen in many sporadic tumors.  The authors proposed that, together with these and their previous findings, that haploinsufficiency plays a crucial role in shaping the cancer genome.

Hemizygosity and Haploinsufficiency

Below are a few definitions from Wikipedia:

Zygosity is the degree of similarity of the alleles for a trait in an organism.

Most eukaryotes have two matching sets of chromosomes; that is, they are diploid. Diploid organisms have the same loci on each of their two sets of homologous chromosomes, except that the sequences at these loci may differ between the two chromosomes in a matching pair and that a few chromosomes may be mismatched as part of a chromosomal sex-determination system. If both alleles of a diploid organism are the same, the organism is homozygous at that locus. If they are different, the organism is heterozygous at that locus. If one allele is missing, it is hemizygous, and, if both alleles are missing, it is nullizygous.

Haploinsufficiency occurs when a diploid organism has only a single functional copy of a gene (with the other copy inactivated by mutation) and the single functional copy does not produce enough of a gene product (typically a protein) to bring about a wild-type condition, leading to an abnormal or diseased state. It is responsible for some but not all autosomal dominant disorders.

Al Knudsen and The “Two-Hit Hypothesis” of Cancer

Excerpt from a Scientist article by Eugene Russo about Dr. Knudson’s Two hit Hypothesis;

for full article please follow the link http://www.the-scientist.com/?articles.view/articleNo/19649/title/-Two-Hit–Hypothesis/

The “two-hit” hypothesis was, according to many, among the more significant milestones in that rapid evolution of biomedical science. The theory explains the relationship between the hereditary and nonhereditary, or sporadic, forms of retinoblastoma, a rare cancer affecting one in 20,000 children. Years prior to the age of gene cloning, Knudson’s 1971 paper proposed that individuals will develop cancer of the retina if they either inherit one mutated retinoblastoma (Rb) gene and incur a second mutation (possibly environmentally induced) after conception, or if they incur two mutations or hits after conception.3 If only one Rb gene functions normally, the cancer is suppressed. Knudson dubbed these preventive genes anti-oncogenes; other scientists renamed them tumor suppressors.

When first introduced, the “two-hit” hypothesis garnered more interest from geneticists than from cancer researchers. Cancer researchers thought “even if it’s right, it may not have much significance for the world of cancer,” Knudson recalls. “But I had been taught from the early days that very often we learn fundamental things from unusual cases.” Knudson’s initial motivation for the model: a desire to understand the relationship between nonhereditary forms of cancer and the much rarer hereditary forms. He also hoped to elucidate the mechanism by which common cancers, such as those of the breast, stomach, and colon, become more prevalent with age.

According to the then-accepted somatic mutation theory, the more mutations, the greater the risk of cancer. But this didn’t jibe with Knudson’s own studies on childhood cancers, which suggested that, in the case of cancers such as retinoblastoma, disease onset peaks in early childhood. Knudson set out to determine the smallest number of cancer-inducing events necessary to cause cancer and the role of these events in hereditary vs. nonhereditary cancers. Based on existing data on cancer cases and some mathematical deduction, Knudson came up with the “two-hit” hypothesis.

Not until 1986, when researchers at the Whitehead Institute for Biomedical Research in Cambridge, Mass., cloned the Rb gene, would there be solid evidence to back up Knudson’s pathogenesis paradigm.4 “Even with the cloning of the gene, it wasn’t clear how general it would be,” says Knudson. There are, it turns out, several two-hit lesions, including polyposis, neurofibromitosis, and basal cell carcinoma syndrome. Other cancers show only some correspondence with the two-hit model. In the case of Wilm’s tumor, for example, the model accounts for about 15 percent of the cancer incidence; the remaining cases seem to be more complicated.

knudsonTwoHit1600

His seminal paper on the two-hit hypothesis[1]

A.G. Knudson, “Mutation and cancer: statistical study of retinoblastoma,” Proceedings of the National Academy of Sciences, 68:820-3, 1971.

The two hit hypothesis proposed by A.G. Knudson.  A description with video of Dr. Knudson talk at AACR can be found at the following link (photo creditied to A.G. Knudson and Fox Chase Cancer Center at the following link:http://www.fccc.edu/research/research-awards/knudson/index.html

Sources

1.            Knudson AG, Jr.: Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Sciences of the United States of America 1971, 68(4):820-823.

2.            Solimini NL, Xu Q, Mermel CH, Liang AC, Schlabach MR, Luo J, Burrows AE, Anselmo AN, Bredemeyer AL, Li MZ et al: Recurrent hemizygous deletions in cancers may optimize proliferative potential. Science 2012, 337(6090):104-109.

3.            Davoli T, Xu Andrew W, Mengwasser Kristen E, Sack Laura M, Yoon John C, Park Peter J, Elledge Stephen J: Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns and Shape the Cancer Genome. Cell 2013, 155(4):948-962.

Other papers on this site on CANCER and MUTATION include:

Cancer Mutations Across the Landscape

Salivary Gland Cancer – Adenoid Cystic Carcinoma: Mutation Patterns: Exome- and Genome-Sequencing @ Memorial Sloan-Kettering Cancer Center

Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

Breast Cancer and Mitochondrial Mutations

Winning Over Cancer Progression: New Oncology Drugs to Suppress Passengers Mutations vs. Driver Mutations

Hold on. Mutations in Cancer do good.

Rewriting the Mathematics of Tumor Growth; Teams Use Math Models to Sort Drivers from Passengers

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

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Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity from the Oncology Pharma forum on LinkedIn

Curator and Writer: Stephen J. Williams, Ph.D.

In an earlier post entitled “Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing” the heterogenic nature of solid tumors was discussed.  There resulted an excellent discussion in the Oncology Pharma forum on LinkedIn so I curated the comments (below article) to foster further discussion. To summarize the original post, this was a discussion of Dr. Charles Swanton’s paper[1] in which he and colleagues had noticed that individual biopsies from primary renal tumors displayed a variety of mutations of the same and different tumor suppressor genes (TSG), thereby not only revealing the heterogeneity of individual tumors but also how tumors can evolve.  Thus it was suggested that individual cells of a primary tumor can represent individual clones, each evolving on a distinct pathway to tumorigenicity and metastasis as each clone would have accumulated different passenger mutations.  It is these passenger mutations which have been posited to be responsible for a tumor’s continued growth (as discussed in the following post Rewriting the Mathematics of Tumor Growth; Teams Use Math Models to Sort Drivers from Passengers).  Indeed, as Dr. Swanton mentioned in the posting that it is very likely a solid tumor contains discrete clones with different driver and passenger mutations and possibly different mutated TSG but also this intra-tumor heterogeneity would have great implications for personalized chemotherapeutic strategies, not only against the primary tumor but against resistant outgrowth clones, and to the metastatic disease, as Swanton and colleagues had found that the metastatic disease displayed tremendously increased genomic instability than the underlying primary disease.

Therefore it may behoove the clinical oncologist to view solid tumors as a collection of multiple clones, each having their own mutagenic spectrum and tumorigenic phenotype.  Each of these clones may acquire further mutations which provide growth advantage over other clones in the early primary tumor.  In addition, branched evolution of a clone most likely depends more on genomic instability and epigenetic factors than on solely somatic mutation.

This is echoed in a  report in Carcinogenesis back in 2005[3] Lorena Losi, Benedicte Baisse, Hanifa Bouzourene and Jean Benhatter had shown some similar results in colorectal cancer as their abstract described:

“In primary colorectal cancers (CRCs), intratumoral genetic heterogeneity was more often observed in early than in advanced stages, at 90 and 67%, respectively. All but one of the advanced CRCs were composed of one predominant clone and other minor clones, whereas no predominant clone has been identified in half of the early cancers. A reduction of the intratumoral genetic heterogeneity for point mutations and a relative stability of the heterogeneity for allelic losses indicate that, during the progression of CRC, clonal selection and chromosome instability continue, while an increase cannot be proven.”

Therefore if a tumor had evolved in time closer to the initial driver mutation multiple therapies may be warranted while tumors which had not yet evolved much from their driver mutation may be tackled with an agent directed against that driver, hence the branched evolution as shown in the following diagram:

branced chain evolution cancer

Cancer Sequencing

Unravels clonal evolution.

From Carlos Caldas. (2012).

Nature Biotechnology V.30

pp 405-410.[2] used with

permission.

 

 

 

 

 

 

 

An article written by Drs. Andrei Krivtsov and Scott Armstron entitled “Can One Cell Influence Cancer Heterogeneity”[4] commented on a study by Friedman-Morvinski[5] in Inder Verma’s laboratory discussed how genetic lesions can revert differentiated neorons and glial cells to an undifferentiated state [an important phenotype in development of glioblastoma multiforme].

In particular it is discussed that epigenetic state of the transformed cell may contribute to the heterogeneity of the resultant tumor.  Indeed many investigators (initially discovered and proposed by Dr. Beatrice Mintz of the Institute for Cancer Research, later to be named the Fox Chase Cancer Center) show the cellular microenvironment influences transformation and tumor development[6-8].

Briefly the Friedman-Morvinski study used intra-cerebral ventricular (ICV) injection of lentivirus to introduce oncogenes within the CNS and produced tumors of multiple cell origins including neuronal and glial cell origin (neuroblastoma and glioma).  The important takeaway was differentiated somatic cells which acquire genetic lesions can transform to form multiple tumor types.  As the authors state, “cellular differentiation and specialization are accompanied by gradual changes in epigenetic programs” and that “the cell of origin may influence the epigenetic state of the tumor”.   In essence this means that the success of therapy may depend on the cellular state (whether stem cell, progenitor cell, or differentiated specialized cell) at time of transformation.  In other words tumors arising from cells with an epigenetic state seen in stem cells would be more resistant to therapy unless given an epigenetic therapy, such as azacytididne, retinoic acid or HDAC inhibitors.

 

So as the Oncology Pharma forum on LinkedIn was such an excellent discussion I would like to post the comments for curation purposes and foster further discussion.  I would like to thank everyone’s great comments below.  I would especially like to thank Dr. Emanuel Petricoin from George Mason and Dr. David Anderson for supplying extra papers which will be the subject of a future post. I had curated each comment with inserted LIVE LINKS to make it easier to refer to a paper and/or company mentioned in the comment.

The comments seemed to center on three main themes:

  1. 1.      Clinicians pondering the benefit to mutational spectrum analysis to determine personalized therapy and develop biomarkers of early disease
  2. 2.      A shift in the clinicians paradigm of cancer development, diagnoses, and treatment from strictly histologic evaluation to a genetic and altered cellular pathway view
  3. 3.      Use of proteomics, microarray and epigenetics as an alternative to mutational analysis to determine aberrant cellular networks in various stages of tumor development

 

Victor Levenson • Thanks for posting this! To be honest, I am puzzled by the insistence on sequencing as a tool for tumor analysis – we know that expression patterns rather than mutations in a limited number of genes determine tumor physiology (or, even more, physiology of any tissue). Since the AACR-2012 we know that different tumors have similar or even identical mutations, so >functional< rather than >structural< differences are important. Frankly, I’d be much more excited learning about expression pattern heterogeneity in tumors.Granted that is much more challenging than NGS sequencing, but the value of the data would be incomparable, especially in its application to biomarker development.

Stephen J. Williams, Ph.D. • Dear Dr. Levenson, thanks for your comments. I agree with you and in no way am insisting on the releiance of sequencing mutations in cancer as the sole means for determining therapy. It is extremely true that tumors will show tremendous heterogeneity of mRNA expression. There are a number of studies (one which I will post on pharmaceuticalintelligence.com) that individual tumor cells will have differing expression patterns based on the levels of regional hypoxia within the tumor as well as other microenvironmental factors. I do have two posts on pharmaceuticalintelligence.com on this matter, curating various programs around the world which are using microarray expression analysis of tumors to determine personalized strategies. I believe the reliance on mutational analysis is based on the drugs that have been developed (such as Gleevec and crizotinib) which are based on mutant forms of BCR-Abl and ALK, respectively. However (as per two posts I did based on Mike Martin on our site “Mathematical Models of Driver and Passenger mutations) where he discusses how certain driver mutations will get the senescent cell over the hump to get to fully transformed and contribute to a certain level of growth while subsequent passengers are responsible for the sustained survival and expansion of the tumor.

Victor Levenson • Dr. Williams, thanks for the comments. Driving a senescent cell into proliferative stage is a tremendous change, which >may< begin with a mutation, but involves dramatic restructuring of transcription patterns that will drive the process. Hypoxia will definitely contribute to variations in the patterns, although will probably not be the main driver of the process. As to whether a mutation or a change in transcription pattern initiate the process, I am not sure we will ever be able to determine <grin>.

Vanisree Staniforth • Thanks for posting! Certainly a thought provoking article with regard to the future of personalized cancer therapies.

 

Dr. Raj Batra • If we follow Dr Levenson’s proposed conceptual approach (which we also published in 2009 and 2010), we are MUCH more likely to significantly impact tumor morbidity and mortality.

Stephen J. Williams, Ph.D. • Thanks Vanisiree and Dr. Batra for your comments. Hopefully we will see, from the future cancer statistics, how personlized therapy have improved outcomes for the solid tumors, like the hematologic cancers. 26 days ago

Emanuel Petricoin • The issue about intra and inter tumor heterogeneity is very important however since it is unknown which mutations are true drivers, an explanation of the results found in these studies simply could be the variances are all in the inconsequential mutations and the commonality is the driver mutations. Moreover, at the end of the day, its not the mRNA expression that we really care about but the functional protein signaling -phosphoprotein driven signaling architecture, that we care about since these are the drug targets directly.

Mohammad Azhar Aziz,PhD • This article addresses the potential complexity of dealing with cancer which is apparently increasing proportionally with the amount of data generated. Intratumor heterogeneity will remain there and even multiple biopsies that are randomly chosen will offer no conclusive solution.Mutations,expression profiles and functional protein signaling (as discussed above) alone can not provide any breakthrough. It will be a composite picture of all these and many other components (e.g. microenvironment, alternative splicing, epigenetics,non-coding RNAs etc.) that will hold the promises in the future. We have made phenomenal advances in understanding each of these aspects separately but definitely lack the tools to integrate all these. Developing tools to integrate all these data may provide some breakthrough in understanding and thus treating cancer.

Emanuel Petricoin • I agree Mohammad in a systems biology approach however the current compendium of drugs largely are kinase inhibitors or enzymatic inhibitors. Since most studies have shown little correlation between gene mutation and protein levels and phosphoprotein levels, for example, it is no wonder why the recent spate of failed trials (e.g. stratification by PIK3CA mutation or PTEN mutation for AKT-mTOR inhibitors) should come as any shock. We will be publishing work using protein pathway activation mapping coupled to laser dissection of a number of intra and inter tumoral analysis that indicates that the signaling architecture appears much more stable.

Stephen J. Williams, Ph.D. • Thank you Dr. Pettricoin for your comments. I eagerly await the publication of your results concerning proteomic evaluation of multiple biopsies of a tumor. I am very interested that you found limited intratuoral heterogeneity of signaling pathways given the diversity of intratumoral microenvironmental stresses (changes in regional hypoxia, blood flow, and populations of cancer stem cells). I agree with you and Mohammed that proteomic profiling will be imperative in determining personalized approaches for targeted therapy. Dr. Swanton had informed me that they had used IHC to determine if mTOR signaling had correlated with the mutational spectrum they had seen. In addition he had mentioned that there was enhanced genomic instability in the metastatic disease relative to the primary tumor and it would be very interesting to see how signaling pathways change in cohorts of matched metastatic and primary tumors. A few years ago we were looking at genes which were completely lost upon transformation of ovarian epithelial cells and worked up one of those genes (CRBP1) in cohorts of human ovarian cancer samples, using expression analysis in conjunction with laser capture microdissection and backed up by IHC analysis, and found that the expression pattern of CRBP1 was uniform in a tumor, either there was a complete loss in all cells in a tumor of CRBP1 or all the cells expressed the protein. Therefore I am curious if intratumor heterogeneity is dependent on the cell lineage and evolution of the transformed cell into a full tumor or a function of a discrete population of stem cells with varied genomic instability. Your results might suggest a more clonal evolution rather than a branched evolution which was found in this paper.
It is interesting that you mention the tough trials with the PTEN/PI3K/AKT axis of inhibitors. In high grade serous ovarian cancer we were never able to find any PI3K, PTEN, nor AKT mutations yet PI3K activity is usually overactive. If feel both your and Mohammed’s assessment that a systems biology approach instead of just relying on DNA mutational analysis will be more important in the future. In addition, there is nice work from Dr. Jefferey Peterson at Fox Chase and the development of a database of kinase inhibitors and activity effects on the kinome, showing the vast amount of crosstalk between once thought linear enzyme systems. If TKI’s will be the brunt of pharma’s development I feel they need to quickly develop as many TKI’s as they can now before we get to a clinical problem (resistance and lack of available therapeutics).

Emanuel Petricoin • Thanks Steven- yes, we are working with Charlie Swanton and Marco on the renal sets- our other studies are from breast and colon cancers. I think one of the things we do that really no one else is doing, unfortunately, is to laser capture microdissect the tumor cells from these specimens so that we have a more pure and accurate view of the signaling architecture. One confounder from the proteomic stand-point is the fact that pre-analytical variables such as post-excision delay times where the tissue is a hypoxic wound and signaling changes fluctuating as the tissue reacts to the ex-vivo condition can really effect things. When we look at tissue sets where the tissue is biopsied and immediately frozen we really dont see big differences in the signaling – the within tumor architecture is much more similar then between. We use the reverse phase array technology we invented to provide quantitative analysis on hundreds of phosphoproteins at once – so a nice view of the functional protein activation network. Your results of CRBP1 in ovarian tumors and the IHC data are very interesting. We will see how this all plays out. Of course once other confounder with the mutational data is that we really dont know what are the drivers and what are the passengers…
Yes I know Jeff Peterson’s work- its fantastic. In the end the hope I think- and in my personal opinion- will be rationally combined therapeutics based on the signaling architecture of each individual patient.

Incidentally, we just published a paper that you may be interested in from a “systems biology” standpoint-

SYSTEMS ANALYSIS OF THE NCI-60 CANCER CELL LINES BY ALIGNMENT OF PROTEIN PATHWAY ACTIVATION MODULES WITH “-OMIC” DATA FIELDS AND THERAPEUTIC RESPONSE SIGNATURES.

Federici G, Gao X, Slawek J, Arodz T, Shitaye A, Wulfkuhle JD, De Maria R, Liotta LA, Petricoin EF 3rd. Mol Cancer Res. 2013 May

also- we published a paper that speaks directly to your point where we compared the signaling network activation of patient-matched primary colorectal cancers and synchronous liver mets. indeed there is huge systemic differences in the liver metastasis compared to the primary. there is no doubt in my mind that we will need to biopsy the metastasis to know how to treat. Looking at the primary tumor as a guide for therapy is a fools errand. here is the paper reference:

Protein pathway activation mapping of colorectal metastatic progression reveals metastasis-specific network alterations.

Silvestri A, Calvert V, Belluco C, Lipsky M, De Maria R, Deng J, Colombatti A, De Marchi F, Nitti D, Mammano E, Liotta L, Petricoin E, Pierobon M.

Clin Exp Metastasis. 2013 Mar;30(3):309-16. doi: 10.1007/s10585-012-9538-5. Epub 2012 Sep 29.

Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd., Manassas, VA, 20110, USA.

Abstract

The mechanism by which tissue microecology influences invasion and metastasis is largely unknown. Recent studies have indicated differences in the molecular architecture of the metastatic lesion compared to the primary tumor, however, systemic analysis of the alterations within the activated protein signaling network has not been described. Using laser capture microdissection, protein microarray technology, and a unique specimen collection of 34 matched primary colorectal cancers (CRC) and synchronous hepatic metastasis, the quantitative measurement of the total and activated/phosphorylated levels of 86 key signaling proteins was performed. Activation of the EGFR-PDGFR-cKIT network, in addition to PI3K/AKT pathway, was found uniquely activated in the hepatic metastatic lesions compared to the matched primary tumors. If validated in larger study sets, these findings may have potential clinical relevance since many of these activated signaling proteins are current targets for molecularly targeted therapeutics. Thus, these findings could lead to liver metastasis specific molecular therapies for CRC.

Adrian Anghel • I think both patterns (protein phosphorylation and mRNA) should be important in this complicated equation of heterogeneity. Let’s not forget the so-called functional miRNA-mRNA regulatory modules (FMRMs). Also I think we have different patterns of this heterogeneity for different evolutive stages of the tumour.

 

Alvin L. Beers, Jr., M.D. • This is a great study, but bad news for attempting to tailor treatment based on molecular markers. Dr. Swanton’s comment: “herterogeneity is likely to complicate matters” is an understatement. Intratumoral heterogeneity, branched, instead of linear, evolution of mutational events portends a nightmare in trying to predict location and volume of biopsies. I am reminded of a series of articles in Nature 491 (22 November 2012) “Physical Scientists take on Cancer”. There is a great comment by Jennie Dusheck: “Cancer researchers now recognize that taming wild cancer cells – populations of cells that evolve, cooperate, and roam freely through the body-demand a wider-angle view than molecular biology has been able to offer. Cross-disciplinary collaborations can approach cancer a greater spatial and temporal scales, using mathematical methods more typical of engineering, physics, ecology and evolutionary biology. The sense of failure so evident five years ago is giving way to the excitement of a productive intellectual partnership.” I’m not certain how well the “productive partnership” is going, but this Swanton study confirms the limitations of molecular biology.

Stephen J. Williams, Ph.D. • Thanks Dr. Beers for adding in your comment and adding in Jennie’s comment. Certainly it is something to be aware of if a cancer center’s strategy is to rely solely on gene arrays to genotype tumors. I think Dr. Pettricoin’s work on using proteomics might give some resolution to the matter however, in communicating with Dr. Swanton, I did not get the feeling of an “all hope is lost” but just that, in the case of solid tumors like renal, that careful monitoring of tumors after treatment may be warranted and, more interestingly, from a scientific standpoint, is the genetic complexity surrounding the origin of the disease, and not simple mutational spectrum of a single clone.

Burke Lillian • This is clinically a very important issue. Right now, sequencing or massive approaches such as pan-phosphorylation studies are helpful because, although we know many of the drivers, these studies are actually identifying new genes or new pathways that are activated. After a few (or several years), we truly will know which genes are typically activated and there will be panels to look for these.

Emanuel Petricoin • yes, I agree. In fact, the company that I co-founded, Theranostics Health, Inc– is launching a CLIA based protein pathway activation mapping test at ASCO that measures actionable drug targets (e.g. phospho HER2, EGFR, HER3, AKT, ERK, JAK, STAT, p70S6) and total HER2, EGFR, HER3 and PTEN. So these tests are coming even now.

 

Alvin L. Beers, Jr., M.D. • I do not think that “all hope is lost” nor did I have the impression that Dr. Swanton feels that way with regards to molecular profiling of cancer. I certainly applaud further research into the molecular aspects of cancer biology. But I do not believe that this will be sufficient. Integrating physicial sciences into cancer biology makes perfect sense toward better understanding of this complex disease.

Eleni Papadopoulos-Bergquist • I have enjoyed reading these comments and different ideas regarding genetic testing and profiling. As a nurse and researcher at heart, this is information that will make a huge impact on drug protocols, therefore allowing the best and most specific treatment to each individual rather than having a standard treatment protocol. Even with the scientific complexity of specifying genotypes of particular cancers, there is still the question of each individuals body responding to treatment. I’d love to have some dialogue regarding immune response.

Bradford Graves • I too have enjoyed reading this discussion. I am not a clinician but as a drug discovery researcher I have been struck by some parallels to the concept of virus fitness in virology – particularly as applied to HIV. Drug discovery cannot wait for the final answers to the many important questions being addressed in the discussion initiated by Dr. Williams. The best we can do is to pursue a broad range of therapeutics that will give the clinicians the armament they will need to either cure a given cancer or to at least turn it into a chronic as opposed to an acute disease. There has been a measure of success in the HIV field and it seems like it will be achievable for cancer. Obviously, to the extent that the labels of driver and passenger mutations can be correctly applied will help to prioritize the targets we address.

David W. Anderson • I would suggest that you look at the following publications:

Horn and Pao, (2009) JCO 26: 4232-4234.

Bunn and Doebele (2011) JCO:29:1-3

Boguski et al. (2009) Customized care 2020: how medical sequencing and network biology will enable personalized medicine. F1000 Bio Report 1:7.

Jones, S et al. (2010). Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors. Genome Biology. 11:R82. Marco Marra’s group in Toronto.

Also look at how companies and organizations like Foundation Medicine, Caris, Clarient, and CollabRx who are using genomics and sequencing on a large scale to address cancer from a personalized/individual approach.

Cancer is/will be a chronic disease requiring individualized/combinatorial therapies in many cases.

Alvin L. Beers, Jr., M.D. • David. These are excellent articles by Paul Bunn and Mark Boguski regarding integrating molecular markers into diagnostic evaluation, and I’ve seen other papers of similiar elk, and likely there will be more to come. Particularly in NSC lung cancer, the SOC is to use these markers up front. Diagnosis based on histology alone can no longer be recommended. The challenge for the future is how to integrate other aspects of cell biology with these markers. It remains daunting that not only do we see heterogeneity in molecular within tumors at a particularly point in time, but that there is often an evolution of markers over time, ie, a “plasticity” of markers, whether treatment is given or not. We know that targeted agents, TKI’s, enzyme inhibitors are not curative, but do give an improvement in PFS. A great deal of this resistance has to do with this “moving target” aspect of cancer cell biology..

 

References:

1.         Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P et al: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. The New England journal of medicine 2012, 366(10):883-892.

2.         Caldas C: Cancer sequencing unravels clonal evolution. Nature biotechnology 2012, 30(5):408-410.

3.         Losi L, Baisse B, Bouzourene H, Benhattar J: Evolution of intratumoral genetic heterogeneity during colorectal cancer progression. Carcinogenesis 2005, 26(5):916-922.

4.         Krivtsov AV, Armstrong SA: Cancer. Can one cell influence cancer heterogeneity? Science 2012, 338(6110):1035-1036.

5.         Friedmann-Morvinski D, Bushong EA, Ke E, Soda Y, Marumoto T, Singer O, Ellisman MH, Verma IM: Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. Science 2012, 338(6110):1080-1084.

6.         Mintz B, Cronmiller C: Normal blood cells of anemic genotype in teratocarcinoma-derived mosaic mice. Proceedings of the National Academy of Sciences of the United States of America 1978, 75(12):6247-6251.

7.         Watanabe T, Dewey MJ, Mintz B: Teratocarcinoma cells as vehicles for introducing specific mutant mitochondrial genes into mice. Proceedings of the National Academy of Sciences of the United States of America 1978, 75(10):5113-5117.

8.         Mintz B, Cronmiller C, Custer RP: Somatic cell origin of teratocarcinomas. Proceedings of the National Academy of Sciences of the United States of America 1978, 75(6):2834-2838.

 

 

Other articles on this site on “PERSONALIZED MEDICINE” and “CANCER” and “OMICS” include:

Personalized medicine-based diagnostic test for NSCLC

Personalized medicine and Colon cancer

Helping Physicians identify Gene-Drug Interactions for Treatment Decisions: New ‘CLIPMERGE’ program – Personalized Medicine @ The Mount Sinai Medical Center

Systems Diagnostics – Real Personalized Medicine: David de Graaf, PhD, CEO, Selventa Inc.

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

Personalized Medicine: Clinical Aspiration of Microarrays

Understanding the Role of Personalized Medicine

Directions for Genomics in Personalized Medicine

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Rewriting the Mathematics of Tumor Growth; Teams Use Math Models to Sort Drivers from Passengers

Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling

Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression Analysis

Proteomics and Biomarker Discovery

 

 Also please see our upcoming e-book “Genomics Orientations for Individualized Medicine” in our Medical E-book Series at https://pharmaceuticalintelligence.com/biomed-e-books/genomics-orientations-for-personalized-medicine/volume-one-genomics-orientations-for-personalized-medicine/

 

 

 

 

 

 

 

 

 

 

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