Saturday, February 05, 2011

Wnt pathway and cancer stem cells.

One of the problems in being retired is that medicine moves on. New techniques, new disciplines and new pathways keep appearing until scientific papers become increasingly opaque. The Wnt genes have been around a little while, but because they have hardly impacted on CLL I have ignored them. However, the last CLL paper I looked at mentioned them so I thought that I ought to try and understand them.

The name Wnt was coined as a combination of Wg (wingless) and Int and is usually pronounced as 'wint'. The wingless gene had originally been identified as a recessive mutation affecting wing and haltere development in the fruit fly, Drosophila melanogaster (halteres are small knobbed structures modified from the hind wings in some two-winged insects; they are flapped rapidly and function as accelerometers to help the insect maintain stability in flight). They can be seen on this picture of a crane fly.
It was subsequently characterized as segment polarity gene (genes whose function is to define the anterior and posterior polarities within each embryonic parasegment) in Drosophila melanogaster that functions during embryogenesis and also during adult limb formation during metamorphosis. The INT genes were originally identified as vertebrate genes near several integration sites of mouse mammary tumor virus (MMTV). The Int-1 gene and the wingless gene were found to be homologous, as shown by similar amino acid sequences of their encoded proteins.

Mutations of the wingless gene in the fruit fly were found in wingless flies, while tumors caused by MMTV were found to have copies of the virus integrated into the genome forcing overproduction of one of several Wnt genes. The effort to understand how similar genes produce such different effects has revealed that Wnts are a major class of secreted morphogenic ligands of profound importance in establishing the pattern of development in the bodies of all multicellular organisms studied.

Wnt proteins are therefore known as secreted morphogens that are required for basic developmental processes, such as cell-fate specification(ie what happens to individual cells in development), progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. There are at least three different Wnt pathways (see figure which will enlarge when clicked):
the canonical pathway, the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway. In the canonical Wnt pathway, the major effect of Wnt ligand binding to its receptor is the stabilization of cytoplasmic beta-catenin through inhibition of the bea-catenin degradation complex. Beta-catenin is then free to enter the nucleus and activate Wnt-regulated genes through its interaction with TCF (T-cell factor) family transcription factors and concomitant recruitment of coactivators. Planar cell polarity (PCP) signaling leads to the activation of the small GTPases RHOA (RAS homologue gene-family member A) and RAC1, which activate the stress kinase JNK (Jun N-terminal kinase) and ROCK (RHO-associated coiled-coil-containing protein kinase 1) and leads to remodelling of the cytoskeleton and changes in cell adhesion and motility. WNT-Ca2+ signalling is mediated through G proteins and phospholipases and leads to transient increases in cytoplasmic free calcium that subsequently activate the kinase PKC (protein kinase C) and CAMKII (calcium calmodulin mediated kinase II) and the phosphatase calcineurin.

In the canonical pathway Wnt proteins bind to cell-surface receptors of the Frizzled (FRZ) family, causing the receptors to activate Dishevelled family proteins (Frizzled and Dishevelled are obviously descriptions of fruit flies when these genes are missing) and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus. Cell surface FRZ proteins usually interact with a transmembrane protein called LRP. LRP binds FRZ, Wnt and axin and may stabilize a Wnt/FRZ/LRP/Dishevelled/axin complex at the cell surface (known as the receptor complex). Phosphorylation of the cytoplasmic domain of LRP by CK1 and GSK3 can regulate axin binding to LRP (interaction 1 in Figure). The protein kinase activity of GSK3 appears to be important for both the formation of the membrane-associated Wnt/FRZ/LRP/DSH/Axin complex and the function of the Axin/APC/GSK3/β-catenin complex. β-catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression (interaction 2, Figure).

Wnt binding, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC (Figure) (APC stands for adenomatous polyposis coli, one of the genes that is involved in bowel cancer). The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. Several protein kinases and protein phosphatases have been associated with the ability of the cell surface Wnt-activated Wnt receptor complex to bind axin and disassemble the axin/GSK3 complex. The protein kinase activity of GSK3 appears to be important for both the formation of the membrane-associated Wnt/FRZ/LRP/DSH/Axin complex and the function of the Axin/APC/GSK3/β-catenin complex. Phosphorylation of β-catenin by GSK3 leads to the destruction of β-catenin (Figure).

The Wnt pathway is obviously important for morphogenesis and my daughter, who was particularly good at developmental biology, remembers it from her time at Oxford. However, this will not interest most of my readers who will be more concerned with the fact that alterations of Wnts, APC, axin, and TCFs are all associated with carcinogenesis. There are at least 17 members of the Wnt family.

The WNT/ β-catenin pathway is activated by WNT1, WNT3, WNT3a, WNT7a, and WNT8. In the absence of Wnt signaling, β-catenin is destined for destruction. Several genes have now been identified as the target of β-catenin /TCF transcriptional regulation. These include MMP7 (Matrix Metalloproteinase-7), UPAR (Urokinase-type Plasminogen Activator Receptor), CD44, c-Myc, c-Jun, FRA1 (Fos-Related Antigen-1), CcnD1 (Cyclin-D1), PPAR-Delta (Peroxisome Proliferative Activated Receptor-Delta), TCF1 (Transcription Factor-1), Fibronectin , Slug , Gastrin, Cox2 (Cyclooxygenase-2) and the Gamma2 chain of Laminin5. Many of these genes, including CcnD1 and c-Myc have crucial roles in cell growth, proliferation and differentiation, and are inappropriately activated in colon cancer, Burkitt's lymphoma and mantle cell lymphoma as well as many other tumors. c-Myc gene induced by β-catenin may induce expression of p53, and p53 upregulated p21WAF1 and p130/RB2, resulting in growth arrest. Another target of β-catenin is Vimentin, a protein involved in cell migration. Vimentin is a direct target of the β-catenin /TCF transactivation pathway in human breast cancer cells. WNT signaling can prevent apoptosis by up-regulating anti-apoptotic proteins, such as the caspase inhibitor, survivin, and stimulate angiogenesis via up-regulation of VEGF (Vascular Endothelial Growth Factor). Several proteases capable of degrading extracellular matrix, such as Matrilysin/MMP7 and MMP26, as well as cell adhesion molecules such as CD44 and NRCAM (Neuronal Cell Adhesion Molecule) are WNT targets that could aid the tumour cells in invasion and metastasis. The Canonical WNT signaling cascade also regulates the NRSF/REST and ENC1 (Ectodermal-Neural Cortex (with BTB-like domain)-1) genes, thereby controlling the progenitor cells. Cldn1 (Claudin-1) is also involved in the Ctnn-Beta -TCF/LEF signaling pathway, and increased expression of Cldn1 may have some role in colorectal tumorigenesis.

One of the interesting recent developments in cancer has been the recognition of cancer stem cells (CSCs). Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. Last September an investigation was published of reporting an examination of the role of Wnt/β-catenin pathway in regulating the growth and maintenance of colonospheres using the human colon cancer cell line HCT-116. Colonospheres formed by HCT-116 cells show over 80% of the cells to be CD44 positive, compared to ≤ 1% in the corresponding parental cells. Additionally, colonospheres showed reduced membrane bound β-catenin but had increased levels of total β-catenin, cyclin-D1 and c-myc and down regulation of axin-1 and phosphorylated β-catenin. Increased expression of β-catenin was associated with a marked transcriptional activation of TCF/LEF. The latter was greatly decreased following down regulation of β-catenin by the corresponding siRNA.

Cancer stem cells that are resistant to conventional chemotherapy seem to be one possible reason that cancer returns after complete remissions. Speculation suggests that this is true not only for epithelial tissues like the colon, but also for leukemias and lymphomas. It may be that therapy targeted to the Wnt/β-catenin pathway will be effective in eliminating this population.

1 comment:

Tricia said...

Another tour de force. No sign of 'chemo brain' after 17 cycles. Amazing.