Copper and BRAF mutation a review

BRAF kinase is mutated, typically val 600 --> glu (v600E) to induce wide spectrum of cancers. MAPK signaling pathway is essential for tumorigenesis, in which a chain of proteins in the cell that communicate a signal from a receptor on the surface of cell to the DNA in the nucleus of the cell. Phosphorylation of MEK1/2 by BRAF is one among the steps in pathway. By the way, phosphorylated MEK1/2 in turn phosphorylates ERK1/2. In normal cells MAPK pathway is turned off shortly but incase of any mutation it cannot be turned off and leads to uncontrolled proliferation that results in tumorigenesis.  Mutated BRAF (BRAF ^Ｖ600E) along with a conjugate, Cu-MEK1, enhance ERK1/2 phosphorylation which results in tumor growth. MEK1 has great affinity towards copper. So, when it binds to copper, results in high level of P-ERK1/2 (phosphorylated ERK1/2) which further enhance tumor formation. Hence it is essential to suppress copper influx inside the cell. Ctr1 (copper transportor 1) is responsible of transporting copper inside the cell. It is believed that if ctr1 is suppressed which may result in copper deficiency there by MEK1 cannot make conjugate with copper and cannot enhance tumor growth.




Reference: http://www.nature.com/nature/journal/v509/n7501/full/nature13180.html

Review on TAMs, which helps in tumor growth

       The research is about how TAMs (Tumor associated Macrophages) differ from MTMs (Mammary tissue macrophages) functionally and phenotypically. Not all macrophages help tumor growth but TAMs do. It was investigated that how TAM differentiate from its precursors and helps in tumor growth. RBPJ (transcriptional regulator of Notch signaling) helps TAMs. Investigation was done on myeloid cells during cancer progression. Myeloid cells consist of more than 50% of CD45+ cells and population I, II, III cells.  Distinguishing Population I, II, III based on cell surface expression may not be accurate. So it was preferred to distinguish based on transcriptional phenotypes. Finally it was concluded that Population I cells are TAMs. i.e  these cells helps tumor growth.

 

        Ly6C+ CCR2+ inflammatory monocytes contribute to MTMs and lesser extent to TAMs. TAMs are derived from CCR2+ monocyte precursors but require less input from blood compared to MTMs. Higher proliferating capacity of TAMs is  required to find their precursor. TAMs expressed higher levels of Ki67 staining and edU incorporation relative to MTMs. Monocytes could differentiate into TAMs invivo. TAMs differentiation pathway is distinct from MTMs pathway. TAMs are not AAMs (Alternatively Activated Macrophages). It was found that TAMs display a gene expression signature associated with Notch signaling pathway. TAMs depends on Notch signaling but TAM differentiation is not completely abolished in the absence of RBPJ (transcriptional regulator of Notch signaling). The data suggested a specific function of RBPJ dependent TAMs in promoting tumor immune tolerance in part by modulating the CD8+ cell response. It was hypothesized that  control of adaptive immune response by TAMs is one among its role in tumor growth.

 

 

Reference: http://www.ncbi.nlm.nih.gov/pubmed/24812208

Protein Synthesis (Translation, Transcription Process)

A review on Lgr5 Stem cells and intestinal paneth cells

Single Lgr5 stem cells when co cultured with paneth cells form long lived, self organizing crypt-villus organoids in the absence of non epithelial niche cells. To conclude this fact, stem cells and/or paneth cells were seeded in round bottomed plates in 10% matrigel. Matrigel, a culture medium which provides almost similar environment as likely the complex extracellular environment found in many tissues.

Matrigel based culture system containing,

1. EFG - Epidermal Growth Factors

2. Wnt agonists R-spondin 1  -  R-spondin 1 is a secretary protein which interacts with Wnt and activates Wnt signals. (Wnt signals are essential for proliferation).

3. BMP inhibitor Noggin - Noggin is a protein, encoded by NOG gene, binds and inactivates members of signalling proteins BMP (Bone Morphogenetic protein).

Single Lgr5 stem cells autonomously grow into crypt like structures with denovo generated stem cells and paneth cells at their bottom. To conclude that, a confocal cross-sectioning of crypt bottoms of  Lgr5 - EGFP - ires - creERT2 mice revealed distribution of paneth cells and Lgr5 stem cells. At the maximum heterotypic contact (paneth cells - stem cells) and minimum of homotypic contact (paneth cells - paneth cells) or (Lgr5 stem cells - Lgr5 stem cells) were found. The same  contact was observed in the organoid cultures at crypt bottoms. To get a clear idea about crypt, villus etc... refer following Figure.

Paneth cells and/or stem cells seeded in round bottom plates in 10% Matrigel and following were observed,

                                                                                           GFP organiods formation

a). 500 Lgr5 stem cells - 500 Lgr5 stem cells

      (RFP).                                                               -           6.7+/-  3.3% of 10 wells / experiment

b). 500 Paneth cells - 500 paneth cells           -          0% of 10wells / experiment

     (YFP)

c).  500 stem cells - 500 paneth cells              -          76.7 +/- 8.8% of 10 wells / experiment

                                                                       GFP - Green Fluorescent Protein

                                                                       RFP - Red Fluorescent Protein

                                                                       YFP - Yellow Fluorescent Protein

Lgr5 stem cells - sorted from a clonal RFP+ organoid culture

Paneth cells  - sorted from a clonal YFP+ organoid culture

Thus Lgr5 stem cells and paneth cells require physical contact. Indeed Lgr5 stem cells are critically dependent on Notch signals.

Stem cell markers.                

1. Notch 1

2. Olfm4

3. FZD7

4. Cdca7

5. Tnfrsf19

6. Lgr5

Paneth cell Markers

1. Dll4

2. Tgfa

3. Egf

4.Wnt11

5. Wnt3

6. Defa1

7. Lyz1

Among genes, most highly enriched in paneth cells, we noted  Wnt3, Wn11, Egf, Tgfa and notch ligand Dll4. Thus paneth cells provide essential signals for stem cells support. High level expression of Wnt3 was confirmed by in situ hybridization. Indeed, Wnt signalling instructs intestinal cells to adopt a proliferative progenitor phenotype. So, when an Wnt secretion inhibitor (porcupine inhibitor) Iwp1 was added, Organoid was entirely lost and proliferation halted. It can be overcome by exogenous Wnt3A. It was concluded that R spondin 1 acts by amplifying the local response to short - range Wnt produced by paneth cells. Thus only the direct neighbours of paneth cells, the Lgr5 stem cells receive strong Wnt signals which can be further, increased by R-spondin 1.

It was recently observed that stem cells and paneth cells doublets strongly increased  plating  efficiency compared to single stem cells. To investigate that concept using in vivo models, whether paneth cells provide essential support to Lgr5 stem cells, used 3 genetic mouse models for paneth cell loss.

A. Mutation of Gfi1

B.Transgenic expression of diphtheria toxinA4 under the paneth cells - specific cryptdin 2 promoter (CR2 - tox176)

C. Conditional deletion of Sox 9

In crypts of Gfi 1 and CR 2 - tox176  (A and B) paneth cells were seen in decreased number. Number of stem cells were again decreased, coincident with paneth cells.

Thus paneth cells serve as multifunctional guardians of stem cells both by secreting bactericidal products and by providing essential niche signals for organoid formation.

Reference: http://www.ncbi.nlm.nih.gov/pubmed/21113151

Picture Reference  : nature