Wnt Signalling Plays Critical Role Programming Embryonic Development Biology Essay

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1. "Over expression LRP6 co-receptors in Wnt/β-catenin signalling. Proceedings of the National Academy of Science. 107, (11) pp 5136-5141. 2. Wnt signalling plays a critical role in programming embryonic development (ref). In the Canonical Wnt pathway, secreted Wnt ligands bind to frizzled family receptors (FZ) and low-density lipoprotein (LDL) receptor-related protein-6, (LRP6) activating Dishevelled (Dvl). Dvl inactivates "destruction complex." Phosphorylation of LRP6 is induced by Glycogen Synthase Kinase-3 (GSK3) and Casein Kinase Iα (CK1α). Axin interacts with the cytoplasmic tail of LRP-6 which provides a docking site for Axin, causing disruption of the "destruction complex", comprised of Adenomatous Polyposis Coli (APC) Axin, GSK3ß and CK1α. Subsequently "the destruction complex" is unable to phosphorylate β-catenin, leading to its stabilisation and translocation into the nucleus. Nuclear β-catenin interacts with T-cell factor/lymphoid enhancer factor (TCF/LEF), stimulating transcription of Wnt target genes regulating cell differentiation, growth and survival (ref). On the other hand in the absence of a ligand β-catenin, the destruction complex phosphorylates β-catenin and marks it for ubiquitin mediated degradation by proteosomes. This results in the inhibition of Wnt target genes (ref).

3. Deregulation of Wnt/β-catenin signalling is frequently caused by mutations of the intracellular components down the cascade. However, in breast cancer, such mutations are rare. Aberrant activation of Wnt/β-catenin therefore must have its origin up stream of the pathway. Bjorklund et al 2009 associated LRP5Δ with tumor formation. Li Y et al 2004 demonstrated that LRP6 influenced distribution and translocation of β-catenin into the nucleus, encouraging tumourigenesis and cell proliferation. Liu et al 2010, reported over-expression of LRP6 co-receptors in human breast cancers. Significant up regulation of LRP-6 occurred in ER and/or HER2 -ve subtypes of breast cancer and triple negative breast cancer (TNB) (ER/PR/HER2 negative). However LRP5 was not up regulated. Liu et al 2010, silenced expression of LRP6 (LRP6 KD) in breast cancer cells in order to observe the effect of LRP6 down regulation on Wnt/β-catenin signalling. In LRP6 KD cells, free β-catenin pool and Wnt signal were decreased. Moreover the expression of Wnt target genes, Cyclin Dl and c-My were reduced, in comparison to the control (expressing of LRP6). More importantly in LRP6 KD cells, cell proliferation and cell growth was reduced, accompanied by low frequency and small sized colonies of cancer cells. On the other hand Liu et al 2010 generated a shRNA-resistant LRP6 (LRP6-Res) construct, which was transfected into LRP6 KD cells, LRP6-Res was able to reinstate Wnt/β-catenin signalling and tumourigenesis. Active form of β-catenin was introduced into control and LRP6 KD. Active β-catenin up regulation enhanced Wnt/β-catenin signalling and recovered cell growth. LRP6 cDNA was introduced in breast cancer cells expressing limited LRP6. In comparison to control, cells over-expressing LRP6 showed enhanced Wnt signalling and increased growth. Liu et al 2010 generated mice expressing xenografts tumours. A dose of control (expressing LRP6) was injected into one into 1 mouse flank, and LRP6 KD into the other. In LRP6 KD flank, tumours developed significantly slower and were smaller in size. Moreover, a decrease in Wnt/β-catenin signalling and its target gene expression was observed. On the other hand in the control flank, there was accelerated tumour development and large in size. Taken together these observations suggest that over-expression of LRP6 cause's aberrant activation of the Wnt/β-catenin, leading to tumourigenesi in ER and/or HER2 negative subtypes of breast cancer.

4. Liu et al 2010 found that LRP6 was over-expressed in 20-36% of breast cancer, largely in ER and/or HER2 negative and TNB. LRP6 up regulation therefore can be used as a diagnostic marker and as a therapeutic target in patients exhibiting above tumour subtypes. Down regulation of LRP6 receptors in breast cancer, suppressed cell growth. Mesd, LRP6 antagonists inhibited tumour growth in vivo. TNB could benefit from treatment with LRP6 antagonist. Over 90% of colorectal cancers are caused by APC mutations. Application of LRP6 antagonist will intercept inactivation of "the destruction complex" allowing degradation of LRP6. Nuclear translocation β-catenin is inhibited hence c-Myc and cyclin D1 oncogenes remain repressed. Overall there will be a reduction in cell/ tumour growth. Currently TNB cancers are treated with Anthracycline/Taxane based chemotherapy in neoadjuvant settings. LRP6 antagonists can be used to treat TNB and perhaps colorectal cancers.

5 Liu et al 2010, demonstrated when LRP6 was knocked down, Wnt/β-catenin signalling, target gene expression and tumorigenesis were down regulated. Taking this into consideration, it would be ideal to design an inhibitor of the LRP6 co-receptor, diminishing downstream signalling. The LRP6 inhibitor would therefore prevent activation of Dvl, which in turn would allow the activation of the "destruction complex" to phosphorylate β-catenin, target it for polyubiquination and degradation by proteosomes. Prevention of β-catenin stabilisation is critical to suppress its interaction with TCF/LEF, stimulating transcription of Wnt target genes regulating cell differentiation, growth and survival. An ideal compound would be a competitive antagonist which would compete for active sites with Wnt ligands, and therefore has a high affinity for LRP6 receptors. Liu et al 2010 identified Mesd as an antagonist of the LRP6 receptor. Lu W et al 2010, reported that Mesd is a specialized folding chaperone and a universal inhibitor of LRP5 and LRP6 ligands. Mesd in it C-terminal contains a 38 amino acid region which allows interaction with LRP6. Liu et al 2010 generated a transgenic mouse Wnt1 driven tumour model (MMTV-Wnt1 tumour xenograft). In MMTV-Wnt1 mice, 10mg/kg of Mesd were intraperitoneal (IP) administrated. Mesd was able to suppress Wnt1 activated Wnt signalling, which was demonstrated by the reduction in tumour volume formation, free β-catenin and reduced expression of Wnt target genes. Li Y et al 2010 reported Dickkopf 1, is a secreted Wnt signaling antagonist that binds to LRP6 and inhibits the Frizzled-Wnt-LRP6 complex formation in response to Wnts. This molecule could also be used as an antagonist of LRP6 receptors. LRP6 antagonist would be an analogous to Herceptin, thus it can be administrated on a drip into a vein, Anthracycline is administrated in similar manner.

6. According to the FDA (Food and Drug Administration) it takes on average 6.5 years for a drug to undergo screening, preclinical research and development, during this time the lead compound is tested for toxicity and how it is metabolised in cultures and animals. In phase I trials the new compound is examined in healthy individuasl for tolerance and safety, which takes approximately 1.5 years. Phase II takes 2 years, where the drug is tested in patient volunteers for side effects and efficacy. In Phase III, efficacy and adverse effect are further tested, which can take 3.5 years. Finally the drug is reviewed by FDA and the launched to all patients. Therefore it can take ~15 years to release a drug on the market. Phase II and III may take longer as it would be difficult to recruit patient volunteers because TNB is associated with high rate of recurrence and mortality rate.

7. Currently there are no specific chemotherapeutic compounds against TNB (Isakoff 2010). Considering that LRP6 receptors are over expressed in TNB, a LRP6 antagonist could aid with prognosis and treatment. In my opinion a LRP6 antagonist can be used in neo-adjuvant settings and adjuvant settings. If applied in neo-adjuvant setting it could be combined with Anthracycline/Taxane and platinum agents (Isakoff 2010). In my opinion a LRP6 antagonist can be used as monotherapy if patients are intolerant to latter compounds. Santana and Perez 2010, in adjuvant settings recommend administration of doxorubicin plus Cyclophosphamide followed by Paclitaxel. LRP6 antagonist could be combined with these compounds but further analysis would be required to observe drug-drug interactions, which may produce undesirable side effects. Liu et al 2010 showed that over-expression of LRP6 was noted at stage I and increased with stage progression. Arrieta et al 2006 found that neoadjuvant anthracycline chemotherapy was applied to patient at stages IIB (23%) and 77% stages IIIA and IIIB. I believe LRP6 antagonists can be administrated at any stage of the cancer development. However it will be more effective in inhibiting tumorigenesis if applied at early stages of cancer.

8. Liu et al 2010 reported over Wnt signalling was aberrantly activated in 40-60% of breast cancers, LRP6 co-receptor was over-expressed in 20-36% of human breast carcinomas. Authors found that Mesd protein to be an antagonist of LRP6 co-receptor inhibiting aberrant activation of Wnt signalling, stabilisation and translocation of β-catenin in nucleus hence suppression of tumourigenesis in mice. Santana and Perez 2010 reported there was no standard first line drug recommended for patients with metastatic breast cancers. In my opinion the marketing strategy would be to primarily target patient suffering from TNB and colorectal cancers. Furthermore exploit the fact that there isn't currently a standard first line drug. Mesd was found to have high affinity for LRP6 co-receptors, 60% bioavailability and it takes 2 hours to reach the blood stream. In comparison to Dkk1, Mesd has limited side effects. The development of a LRP6 antagonist could potentially return large profits.

9. Liu et al 2010 found administration of Mesd in mice showed no side effects, H&E staining revealed no abnormalities in gastro-intestinal structures and skin. Bodine 2008 reported that mutation or loss of LRP5/6 receptors reduced bone mineral density and fractures. Huelsken et al 2001found that administration of LRP6 antagonist Dickkopf 1for seven days, caused destruction of crypts, villi and glandular structures in colon and small intestine, in adult mouse loss of hair follicles was observed. Liu et al 2010 did not observe such adverse effects in mouse models. Patients may experience general side effects of chemotherapy such as weakness, headache and diarrhoea. Long term inhibition of LRP6 co-receptors may cause osteoporosis, hence longitudinal data collection is required.TNB breast carcinomas lack expression of oestrogen receptors, progesterone receptors, and human epidermal growth factor receptor type 2. LRP6 receptors are up regulated in ER and/or HER2 -ve subtypes of breast cancer, and only this portion of patients will benefit. In mice tumour volume was significantly reduced after 3 weeks of Mesd treatment, therefore I would expect a cycle to last 3-4 weeks, but further research would be required.

10. Li Y et al 2010 reported suppression of Wnt/β-catenin signalling, Dkk1 interacts with LRP6 receptors preventing construction of Fz-Wnt-LRP6 complexes. In comparison to Mesd, Dkk1 has severe side effects. Gluz et al 2009 "poly-ADP-ribose-polymerase-1 inhibitors, agents targeting the epidermal growth factor receptor, multityrosine kinase inhibitors or antiangiogenic agents" are at different clinical phases.