Structural Biochemistry/Role of TGF-β in CML cells

Characteristics & Functions of TGF-β
TGF-β (Transforming growth factor-Beta) is a cytokine that is associated with several different cell functions including cell growth, differentiation, apoptosis and migration. It is an important protein in question because depending on the cellular context, TGF-β can act as an anti-cancer agent or as a promoter of cancer. Understanding it's function in the cell will deepen current understanding of its role in cancers such as Chronic Myeloid Leukemia (CML). TGF-β is known for preventing cell growth in epithelial cells, endothelial cells, hematopoietic cells and lymphocytes. TGF-β was found to produce epithelial-mesenchymal transition (EMT), which encourages the invasion and metastasis of cancerous cells. Because TGF-β also regulates immune activity in cancer cells towards towards later stages, the cytokine is thus understood to both promote and suppress tumor function in various situations. Normal activation of TGF-β prevents uncontrollable growth in cells; however, once affected by oncoproteins, TGF-β plays an important role in the maintenance of cancerous cells. In leukemic cancers specifically, the natural growth inhibitory function and signaling of TGF-β is suppressed by the expression of certain oncoproteins such as "Evil." Another onco-protein, BCR/ABL, found in chronic myeloid leukemia, increases TGF-β production, which subsequently indirectly works to maintain cancer cell growth. It does so through its activation of two intracellular pathways: PI3K/Akt/NF-kB/MMP9 and Akt-FoxO

PI3K/Akt/NF-κB/MMP9 Pathway
On of the pathways activated by TGF-β, PI3K/Akt/NF-κB/MMP9 is responsible for increasing the synthesis of soluble Kit ligand along with intercellular molecule-1. These two end products of the pathway are significantly important in cancer stem cell progression and survival and are found to be activated in cancerous hemangioblasts (multipotent cells capable of differentiating into hematopoietic and endocrine cells). Increased production of intercellular molecule-1 (s-ICAM-1) prevents T lymphocytes and natural killer cells from recognizing the cancerous cell in which it is found. The increased levels of kit ligand promotes cancer cell mobilization to blood vessels more specifically towards the outer parameters of the circulatory system. By up-regulating TGF-β expression, MMP-9 (matrix metalloproteinase-9) levels, and consequently both kit ligand and s-ICAM-1 levels, rise and contribute to cancer cell progression in the body.

Akt-FoxO Pathway
Another pathway activated by TGF-β, the Akt-FoxO pathway is involved with cancer stem cell maintenance. Also found activated in cancerous hemangioblasts, this pathway ensures that the cell retains its stem cell qualities along with its tumor characteristics.

Chronic Myeloid Leukemia and its relationship with TGF-β
Chronic Myeloid Leukemia (CML) is a cancer of white blood cells and is developed by a chromosomal translocation of the chromosomes BCR and ABL to form the BCR/ABL mutated chromosome, also known as the Philadelphia chromosome. This BCR/ABL mutation codes for the BCR/ABL oncoprotein, which is an activated tyrosine kinase and is found in all CML patients. A common treatment for this disease is Imatinib: an ABL tyrosine kinase inhibitor. Introducing this inhibitor into CML hemangioblasts, it was found to also prevent TGF-β1 activation. (TGF-β1 levels are up-regulated in CML whereas TGF-β2 and TGF-β3 levels are normal compared to non-cancerous hemangioblasts). In addition to seeing an increase in TGF-β1 with the treatment of Imatinib, MMP-9, s-Kit ligand and s-ICAM-1 were likewise discovered to be expressed more. This discovery made a clear connection of TGF-β with the BCR/ABL oncoprotein and thus is implied to have a significant effect on CML stem cell survival and growth via its activation of Kit ligand and ICAM-1.

Family TGF-β proteins in Smad & non-Smad pathways
TGF-β is associated with the following group of family proteins: other activins, bone morphogenetic proteins, and other TGF-β proteins. These proteins signal via two different serine-threonine kinases called Type I and Type II. If Type I is activated, it is able to move a TGF-β activated Smad protein into the nucleus in which the protein then regulates the transcription of certain target genes. Non-Smad pathways such as Erk, JNK and p38 MAP are also regulated by TGF-β. Studying both non-smad and smad pathways, scientists may determine whether smad signalling is involved producing MMP-9 in CML hemangioblasts and consequently whether or not it plays a role in cancer stem cell maintenance and survival. On example of how smad signaling contributes to a rise in MMP-9 levels is the HSc025 inhibitor. HSc025 inhibits TGF-β Smad signaling by activating nuclear translocation of y-box binding protein I. This inhibitor fails to prevent TGF-β activation of MMP-9 in the cell. Therefore, studying Smad pathways and non-Smad pathways, scientists may determine how the production of MMP-9 is affected in CML by certain inhibitors.

Non-Smad Pathways in TGF-β activation
In general, Smads are identified as transcription factors that are involved with conveying intracellular responses to TGF-β. While Smad pathways play a clear role in TGF-β activation, recently non-Smad pathways have been discovered to likewise be activated by TGF-β. The details concerning Non-Smad pathways upon activation of TGF-β can be analyzed under several main branches: JNK/p38MAPK kinase pathways, Rho-like GTPase signaling pathways and phosphatidylinositol-3-kinase/AKT pathways. Understanding the biochemical mechanisms and functions of these pathways will solidify and broaden the current understanding of TGF-β multi functional characteristics.

JNK/p38 MAPK pathway
JNK and p38 MAPK are part of a the third layer of a signaling cascade in which they are activated indirectly by TGF-β and directly by MAP kinase kinases (MKKs). JNK is activated by MKK4 while p38 MAPK is activated by MKK3/6. Going back from along the cascade towards TGF-β, TGF-β-activated kinase 1 (TAK1) is responsible for activating MAP3Ks (the activators of MKKs). Expression of TAK1 in cells is important in deciding whether a cell will undergo apoptosis or not. Over expression of TAK1 causes cells to undergo apoptosis while making TAK1 inactive prevents cells from dying. While the TAK1-JNK/p38 MAPK pathway in a non-Smad pathway, its link to TGF-β/BMP induced apoptosis shows that this non-smad pathway participates with smad pathways.

PI3K/Akt pathway
This pathway is another non-smad pathway involved with TGF-β activation that contributes to EMT, cell migration, TGF-β mediated fibroblast proliferation and morphological transformation. Recently, the PI3K/Akt pathway has been found to interfere with smad pathways (specifically Smad3) along with the transcription factor FoxO. This interaction with the Smad3 pathway prevents it from participating in transcription by preventing both TβR1 mediated phosphorylation and nuclear localization of Smad3. Because of this distinct interference with transcription via a smad-pathway, the PI3K/Akt non smad pathway is an important factor in understanding possible mechanisms of TGF-β mediated growth inhibition and transcription effects.

Future research involving TGF-β in CML
Currently one the main treatment for CML, Imatinib does not completely destroy all CML stem cells. One of the reasons for this failure may be tied to the TGF-Β/Akt/FoxO pathway mentioned above. Because this pathway is involved with stem cell maintenance, further research on this pathway may shed some light on how to eradicate CML stem cells without fear of relapse. Inhibiting TGF-β activation or depleting FoxO3a may prove to be a good potential combination with the already existing Imatinib treatment.