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The role of selected microRNAs (miRNAs) in the negative regulation of pediatric retinoblastoma progression
- 1. z The role of selected microRNAs (miRNAs) in the negative regulation of pediatric retinoblastoma progression Ashtyn Zapletal
- 2. zRetinoblastoma Background Information Autosomal dominant retinal cancer in children1,2 9,000 children each year1 Leukocoria, strabismus, and central vision loss are typical presenting symptoms3 Prognosis favorable if caught early and treated with chemotherapy (etoposide, carboplatin, and doxorubicin) or enucleation4 https://www.aao.org/eye-health/diseases/what-is-retinoblastoma 1. Mallipatna A,, et al. Asia-Pacific Journal of Ophthalmology. 2016. 2. Plousiou M,, et al. Front Genet. 2019. 3. Mendoza PR, et al. Progress in Molecular Biology and Translational Science Molecular Biology of Eye Disease. 2015. 4. Fabian ID, et al. Oncogene. 2018
- 3. z Genetics of Retinoblastoma RB1 gene codes for assembly of the retinoblastoma protein, a tumor suppressor gene1 pRb and its function with E2F3 “2 hit hypothesis” explains determinant of risk1 Inherited germline mutation = 1st hit1 Somatic mutation = 2nd hit1 Purely somatic hits also possible.3 1. Mallipatna A,, et al. Asia-Pacific Journal of Ophthalmology. 2016. 3. Mendoza PR, et al. Progress in Molecular Biology and Translational Science Molecular Biology of Eye Disease. 2015.
- 4. z 3. Mendoza PR, et al. Progress in Molecular Biology and Translational Science Molecular Biology of Eye Disease. 2015. * Retinoblastoma protein role in cell cycle regulation
- 5. z The microRNA (miRNA) role in Rb miRNome landscape analysis revealed a 30 miRNA core indicated in Rb progression5 Vast number of miRNAs implicated5: 1. miRNA-188-5p in the Wnt/β-catenin pathway6 2. miRNA-21 in the PI3K/Akt pathway7 3. miRNA-24 interaction with c-Myc8 https://www.genengnews.com 5. Castro-Magdonel BE, et al. BMC Cancer. 2017. 6. Yang M, et al. Onco Targets Ther. 2019. 7. Gui F, et al. Cell Biology International. 2016. 8. Yu F, et al. International Journal of Biological Macromolecules. 2019.
- 6. z z Intersection of pathways https://www.jneurosci.org 5. Castro-Magdonel BE, et al. BMC Cancer. 2017. 6. Yang M, et al. Onco Targets Ther. 2019. 7. Gui F, et al. Cell Biology International. 2016. 8. Yu F, et al. International Journal of Biological Macromolecules. 2019. 9. Persad A, et al. Genes Cancer. 2016 z z Intersection of pathways https://www.jneurosci.org 5. Castro-Magdonel BE, et al. BMC Cancer. 2017. 6. Yang M, et al. Onco Targets Ther. 2019. 7. Gui F, et al. Cell Biology International. 2016. 8. Yu F, et al. International Journal of Biological Macromolecules. 2019. 9. Persad A, et al. Genes Cancer. 2016
- 7. z Inhibition of ID-4 in the Wnt/- catenin pathway DNA-binding inhibitor ID-4 is a transcriptional regulator of the Wnt/- catenin pathway6 miRNA-188-5p overexpression reduces ID4 mRNA levels by binding to the 3’ UTR6 -catenin nuclear translocation increases c-Myc expression6,8 Upregulated c-Myc levels leads to increased viability and invasion8 Potential therapeutic avenue6 6. Yang M, et al. Onco Targets Ther. 2019. 8. Yu F, et al. International Journal of Biological Macromolecules. 2019.
- 8. z 6. Yang M, et al. Onco Targets Ther. 2019.
- 9. z Downregulation of PTEN by miRNA-21 PTEN functions to inhibit PI3K activity, thus increasing Akt, mTOR, and c-Myc activation7,10 miRNA-21 binds to 3’ UTR of PTEN leading to inactivation6,7 An miRNA-21 inhibitor blocks overexpression of miRNA-217 7. Gui F, et al. Cell Biology International. 2016. 6. Yang M, et al. Onco Targets Ther. 2019. 7. Gui F, et al. Cell Biology International. 2016. 10. Bautista SJ, et al. Journal of Biological Chemistry. 2018.
- 10. z z • Relative protein expression levels highlight the effectiveness of the miRNA-21 inhibitor: • Increased PTEN levels7 • Decreased PI3K and Akt7 • Without miRNA-21 inhibition, irregular PI3K/Akt activation leads to c-Myc transcriptions and unwanted cell growth/decreased apoptosis7 7. Gui F, et al. Cell Biology International. 2016. 7. Gui F, et al. Cell Biology International. 2016.
- 11. z c-Myc regulation by miRNA-24 c-Myc is a proto-oncogene constitutively expressed in Rb8 Roles in both mitosis and apoptosis8,11 c-Myc is a direct target of miRNA-24, leading to its deactivation8,11 Proper balance is vital for retinal cell development8 8. Yu F, et al. International Journal of Biological Macromolecules. 2019. 11. Shang Y, et al. Biomedicine and Pharmacotherapy. 2018. 8. Yu F, et al. International Journal of Biological Macromolecules. 2019.
- 12. z miRNA-24 levels are reduced in Rb tissues8 Unchecked c-Myc expression results8 Unimpeded c-Myc leads to elevated mitotic initiation and depressed regulatory cell suicide8,11 A miRNA-24 mimic introduced into Rb cell lines decreased c-Myc expression8 Potential prospects for therapeutic intervention using miRNA-24 activators8 8. Yu F, et al. International Journal of Biological Macromolecules. 2019. 8. Yu F, et al. International Journal of Biological Macromolecules. 2019. 11. Shang Y, et al. Biomedicine and Pharmacotherapy. 2018.
- 13. z Resources: 1. Mallipatna A, Marino M, Singh AD. Genetics of Retinoblastoma. Asia-Pacific Journal of Ophthalmology. 2016;5(4):260-264. doi:10.1097/apo.0000000000000219. 2. Plousiou M, Vannini I. Non-Coding RNAs in Retinoblastoma. Front Genet. 2019;10:1155. Published 2019 Nov 14. doi:10.3389/fgene.2019.01155 3. Mendoza PR, Grossniklaus HE. The Biology of Retinoblastoma. Progress in Molecular Biology and Translational Science Molecular Biology of Eye Disease. July 2015:503-516. doi:10.1016/bs.pmbts.2015.06.012 4. Fabian ID, Onadim Z, Karaa E, et al. The management of retinoblastoma. Oncogene. 2018;37(12):1551-1560. doi:10.1038/s41388-017-0050-x. 5. Castro-Magdonel BE, Orjuela M, Camacho J, et al. miRNome landscape analysis reveals a 30 miRNA core in retinoblastoma. BMC Cancer. 2017;17(1):458. Published 2017 Jul 1. doi:10.1186/s12885-017-3421-3 6. Yang M, Li Y, Wei W. MicroRNA-188-5p Promotes Epithelial-Mesenchymal Transition by Targeting ID4 Through Wnt/β-catenin Signaling in Retinoblastoma. Onco Targets Ther. 2019;12:10251–10262. Published 2019 Nov 27. doi:10.2147/OTT.S229739
- 14. z Resources: 7. Gui F, Hong Z, You Z, Wu H, Zhang Y. MiR-21 inhibitor suppressed the progression of retinoblastoma via the modulation of PTEN/PI3K/AKT pathway. Cell Biology International. 2016;40(12):1294-1302. doi:10.1002/cbin.10678. 8. Yu F, Pang G, Zhao G. ANRIL acts as onco-lncRNA by regulation of microRNA-24/c-Myc, MEK/ERK and Wnt/β-catenin pathway in retinoblastoma. International Journal of Biological Macromolecules. 2019;128:583-592. doi:10.1016/j.ijbiomac.2019.01.157. 9. Persad A, Venkateswaran G, Hao L, et al. Active β-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A. Genes Cancer. 2016;7(11- 12):368–382. doi:10.18632/genesandcancer.128 10.Bautista SJ, Boras I, Vissa A, et al. mTORC1 controls glycogen synthase kinase 3β nuclear localization and function. Journal of Biological Chemistry. June 2018. doi:10.1101/277657. 11.Shang Y. LncRNA THOR acts as a retinoblastoma promoter through enhancing the combination of c-myc mRNA and IGF2BP1 protein. Biomedicine & Pharmacotherapy. 2018;106:1243-1249. doi:10.1016/j.biopha.2018.07.052. Concluding Remarks and Questions
Editor's Notes
- Hello, my name is Ashtyn Zapletal and today I am going to speak on the role of selected microRNAs in the negative regulation of pediatric retinoblastoma progression.
- Retinoblastoma (Rb) is an autosomal dominant retinal cancer usually affecting children ages 15 months – 24 months, counting for roughly 9,000 new cases each year. Children with retinoblastoma often present with leukocoria and strabismus. Ischemic necrosis of the retinal tumor leads to calcification, resulting in the characteristic “white pupil,” AKA leukocoria. Central vision loss can also be seen. Prognosis is favorable if Rb is caught early and treated with chemotherapy agents such as etoposide, carboplatin, and doxorubicin, or enucleation which is removal of the eye. In order to potentially decrease the unwanted side effects associated with chemotherapy and enucleation, research is warranted into further treatments to curb Rb before it progresses into an unfavorable prognosis.
- The RB1 gene is responsible for providing instructions on how to assemble the retinoblastoma protein. This protein is known as a tumor suppressor gene, inhibiting the cell cycle when a mutation has occurred. The function of pRb is to bind to E2F, a transcription factor involved in cell cycle progression, and inactivate it. Consequently, inactive Rb allows for uninterrupted E2F signaling. Given the autosomal dominant nature of retinoblastoma, there is a 50% chance a child of a parent with a RB1 mutation will also inherit the mutation. Knudson’s “2-hit” hypothesis of retinoblastoma states that 2 mutational events are necessary for progression of the disease such as an inherited germline mutation of RB1 and a somatic mutation. This means that children born with an already mutated RB1 gene only need one more mutation to initiate Rb formation. 90% of those with an inherited single RB1 mutation will develop Rb. Purely somatic hits are also possible.
- Here is a graphic representation of Rb role in inhibiting the E2F transcription factor. Although this slide is busy, the focus is where the red star is, showing Rb attached to E2F. When Rb is phosphorylated by CDKs, it is released from E2F and allows the cell cycle to continue. If the retinoblastoma protein is non-functional, unchecked cell cycle progression can occur and tumors can form.
- MicroRNAs, or miRNAs, are small non-coding RNA strands that hybridize to complementary mRNAs and function to silence them by preventing translation or promoting cleavage. Because of this negative regulation, miRNAs are often implicated in carcinogenesis and are of clinical importance for both biomarkers of cancer and potential therapeutic interventions. A miRNome landscape analysis done by Castro-Magdonel revealed a 30 miRNA core indicated in Rb progression. I am highlighting 3 prominent miRNAs that have been implicated, but there are many, many more. To start, miRNA-188-5p binds to and inactivates ID-4 mRNA, leaving the Wnt/-catenin pathway unchecked. miRNA-21 functions to downregulate PTEN to allow for atypical PI3K/Akt signal activation. Finally, miRNa-24 directly interacts with a downstream member common to both pathways, c-Myc. I will now further go into detail about these various pathways. Along the way, I will highlight current research being done and how it directly affects potential therapeutics in the future.
- In this image, you can see the intersection of the pathways. Retinoblastoma development can be advanced through the interaction of the Wnt/-catenin and PTEN/PI3K/Akt pathways. Although the two pathways vastly differ in various mechanisms, a common denominator is the involvement of c-Myc, a proto-oncogene implicated in Rb.8,9 In the Wnt/-catenin signaling pathway, Wnt, a glycoprotein, binds to a Frizzled family receptor (Fz). Upon binding, a destruction complex including glycogen synthase kinase 3 (GSK3) is disrupted. The destruction complex acts to mark -catenin for destruction, keeping a balance between growth and quiescence.6,9 When there is interference of the destruction complex, the -catenin molecule is not degraded and can accumulate within the cell. Build-up of intracellular -catenin initiates translocation into the nucleus and transcription of various proteins. One such protein is c-Myc.6,9 In addition to the Wnt/-catenin pathway, the PTEN/PI3K/Akt pathway includes c-Myc.9 Phosphatase and tensin homolog (PTEN), a tumor suppressor, negatively regulates phosphoinositide 3-kinase (PI3K) signaling involved in cell cycle regulation.7,9 PTEN dephosphorylates phosphatidylinositol triphosphate (PIP3) and prevents protein kinase B (Akt) recruitment to the cell membrane so it can no longer be activated.7,9 Aberrant PI3K signaling activates the Akt-mTOR phosphorylation cascade. This cascade event also leads to the increased production of c-Myc.7,8,9
- The ID4 gene encodes for DNA-binding protein inhibitor ID-4. It acts as a transcriptional regulator, thereby inhibiting unwanted Wnt/-catenin signaling. Overexpression of miRNA-188-5p inhibits ID4 3’-UTR luciferase function and reduces ID4 mRNA levels. Luciferase is used as a detection mechanism for transcriptional activation. The sequence of miRNA-188-5p aligns with the sequence of ID-4 3’UTR and causes its inactivation. Since ID4 functions to suppress retinoblastoma establishment, the negative regulation of miRNA-188-5p on ID-4 mRNA leaves the Wnt/-catenin pathway unchecked and overstimulated, poised for tumor formation. To tie the pathways together, -catenin translocates to the nucleus and increases expression of c-Myc. The up-regulated levels of c-Myc protein lead to increased cell viability, migration, and invasion. C-Myc also functions to decrease apoptosis. Inhibiting miRNA-188-5p could be a potential therapeutic avenue to pursue.
- In a study done by Yang et al. in 2019, miRNA-188-5p was shown to be overexpressed in murine retinoblastoma tissues. In these graphs, red is overexpression. In the study, human cell samples from patients with retinoblastoma were collected and examined for miRNA expression. Additionally, xenographs were generated in rat pups with retinoblastoma-prone cell lines WERI-Rb-1 and Y79 and compared to normal ARPE-19 (human retinal pigment epithelial cell) cell lines. The black boxes are highlighting the 188-5p markers. As can be seen in panel C, there is a statistically significant increase in miRNA-188-5p levels in human Rb tissues compared to normal tissues. In the panels D and E, miRNA 188-5p is increased in both WERI-Rb-1 and Y79 lines as compared to ARPE-19 lines. This meets Yang’s hypothesis that 188-5p overexpression plays a role in retinoblastoma progression via the effects discussed in the previous slide.
- Here we are in the overall scheme. As mentioned previously, PTEN functions to regulate PI3K activity and inhibit further downstream activation of Akt. Without PTEN, Akt becomes phosphorylated and activates the mammalian target of rapamycin (mTOR). mTOR functions as a serine-threonine protein kinase and regulates cell proliferation, transcription, and protein synthesis. Dysregulation in mTOR stimulation allows for continual cell growth within retinoblastoma tumors. mTORc1 prevents nuclear localization of GSK3β. GSK3β plays a role in marking c-Myc for proteasomal degradation. Inactivation of GSK3β by mTORc1 leads to more available c-Myc. Interestingly, increased miRNA-21 levels are present in Rb tissues as compared to normal retinal tissue. MiRNA-21 binds to the 3’-UTR of PTEN and inhibits the luciferase function, thereby displaying the reduction in PTEN activity.
- A miRNA-21 inhibitor has been shown to suppress cell proliferation by blocking the overexpression of miRNA-21 that would ultimately lead to irregular PI3K/Akt activation. In this study done by Gui et al, Gui’s hypothesis was that miRNA-21 overexpression depressed PTEN functioning. In fact, an miRNA-21 inhibitor used here increased expression of PTEN and decreased expression of PI3K and Akt in a statistically significant manner in a murine sample. Irregular PI3K/Akt activation ultimately leads to c-Myc transcription and its unwanted effects of increased cell growth and decreased apoptosis. Further investigation must be done to explore the potential uses of a miRNA-21 inhibitor for retinoblastoma treatment.
- Here is where we are in the grand scheme of things. C-Myc gene codes for the c-Myc protein, that, as explained earlier, functions as a proto-oncogene and is constitutively expressed in cancers, especially Rb. To increase cell growth, c-Myc upregulates cyclins and downregulates p21, two factors important in the initiation and extinction of mitosis, respectively. To decrease apoptosis, c-Myc upregulates Bcl-2, an anti-apoptotic regulator protein. C-Myc mRNA is known to be a direct target of miRNA-24. Mechanistically, miRNA-24 binds to the c-Myc mRNA and leads to its deactivation by binding to the 3’ UTR Proper balance between c-Myc and miRNA-24 is vital for retinal cell formation.
- In retinoblastoma cells, miRNA-24 levels are reduced, resulting in unchecked c-Myc expression. Unimpeded c-Myc expression leads to elevated mitotic initiation and depressed regulatory cell suicide. In this study done by Yu et al, a miRNA-24 mimic was introduced into Y79 Rb cell lines. The hypothesis was that reintroducing miRNA-24 to cells would inhibit c-Myc activation. In panel C, Y79 cells showed decreased c-Myc expression with the miRNA-24 mimic and increased expression with an inhibitor. Panel D shows western blotting results for c-Myc expression as well. Potential prospects for therapeutic intervention using miRNA-24 activators should be further examined.
- In conclusion, the results from these 3 experiments show the importance of further miRNA research, not just with the 3 mentioned miRNAs, but with additional miRNAs that have not been researched before. With 27 more miRNAs left out of the 30 miRNA core that was found, there is seemingly limitless potential for therapeutic avenues. Some limitations include translating mouse studies into human studies, as research done on children carries additional ethical risks. This information is important because it could greatly impact a child’s well-being and provide other avenues of treatment beyond chemotherapy or enucleation, which carry their own limitations and side effects. As more emphasis is placed on miRNA roles in Rb, I believe the clinical impact could be greater than we can even imagine.