Modulation in the expression levels and protein localization of cell volume and actin cytoskeleton associated proteins by the Slit/ Robo signaling pathway in Glioblastoma multiforme.

Presenter Information

Montserrat Roberts
Natanael Zarco
Hugo Guerrero- Cazares

Faculty Sponsor

Dr. Natanael Zarco PhD and Dr. Hugo Guerrero-Cazares MD, PhD

Faculty Sponsor College

College of Arts and Sciences

Faculty Sponsor Department

Biology

Location

SOARS Virtual Conference

Presentation Website

https://unfsoars.domains.unf.edu/2021/posters/modulation-in-the-expression-levels-and-protein-localization-of-cell-volume-and-actin-cytoskeleton-associated-proteins-by-the-slit-robo-signaling-pathway-in-glioblastoma-multiforme/

Keywords

SOARS (Conference) (2021 : University of North Florida) – Archives; SOARS (Conference) (2021 : University of North Florida) – Posters; University of North Florida -- Students -- Research – Posters; University of North Florida. Office of Undergraduate Research; University of North Florida. Graduate School; College students – Research -- Florida – Jacksonville – Posters; University of North Florida – Undergraduates -- Research – Posters; University of North Florida. Department of Biology -- Research – Posters; Honorable Mention Award

Abstract

Honorable Mention Winner

Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor in adults1. Even after surgical resection, chemotherapy, and radiation, the average patient survival rate is 14 months2. Recent studies indicate that GBM tumors that contact the lateral ventricles within the subventricular zone (SVZ) tend to migrate and recur at distal locations after surgical resection3. One important signaling pathway in Neural Progenitor Cells (NPCs) migration from the SVZ to the olfactory bulbs is the Slit/Robo signaling pathway4. Slit2 proteins are produced by the choroid plexus and create a gradient across ependymal cells. Slit2 proteins then interact with the Roundabout (Robo) receptor on NPCs and acts as a chemo-repelling agent5. This Slit2/ Robo1 signaling pathway is a possible mechanism that contributes to GBM migration from the SVZ to distal locations. The intracellular changes when the Slit/Robo pathway is activated includes changes in cell volume regulation and actin cytoskeleton dynamics6. We then examined how the Slit2/ Robo1 signaling pathway causes changes in the expression of cell volume regulatory genes (AQP4, KCC1, NKCC1, NCC), actin cytoskeleton genes regulatory genes (p-ERM, p-Cofilin), and the localization of actin-associated proteins (FAK, Paxillin, N-WASP, Vinculin). It was found that the Slit/Robo signaling pathway increases the expression levels of genes that contribute to cell volume regulation in GBM cells, and the Slit/Robo signaling pathway increases the phosphorylation of p-Cofilin and p-ERM. This indicates that the Slit2/ Robo1 pathway participates in the regulation of cell volume and actin cytoskeleton dynamics in GBM cells.

Comments

Audio Presentation Transcript:

Hello everyone, my name is Montserrat Roberts and my research is on The modulation in the expression levels and protein localization of cell volume and actin cytoskeleton associated proteins by the Slit/Robo signaling pathway in Glioblastoma multiforme.
For ease of presenting this poster in a virtual format, I have taken screen shots of each segment and placed them into a PowerPoint presentation
• To give everyone a brief background of brain tumors in general:
• Primary brain originate from brain tissue and can be categorized as non-gliomas or gliomas
• Non-gliomas can be composed of neurons or any non-glial tissue
• Gliomas are tumors that are composed of glial cells which classified according to the World Health Organization into 4 different grades depending upon how aggressive the tumor is predicted to behave clinically
• Glioblastoma multiforme, is classified as a Grade 4 glioma, and is the most malignant of all brain tumors with a five-year relative survival rate of only 4%2
• The reason for GBM’s rather bleak prognosis3 is that it is highly aggressive and invasive in nature
• Recent studies indicate that GBM tumors that contact the lateral ventricles tend to migrate and recur at distal locations after surgical resection 3,4.
• These patients with LV-proximal tumors have a lower overall survival rate
• We do not know the exact reason as to why LV-proximal tumors recur at distal locations, but a possible explanation is that there is something in the LV or cerebrospinal fluid that contributes to the recurrence of GBM at distal locations
• We do know that neural progenitor cells, which are cells capable of differentiating into neurons or glial cells, migrate to the olfactory bulbs by way of the rostral migratory stream
• The mechanism by which NPCs migrate to the olfactory bulbs is that they migrate away from the lateral ventricles due to a chemorepellent agent
• This chemo repelling agent is known as Slit proteins
• Slit proteins are proteins produced by the choroid plexus and released into the CSF where they create a gradient across ependymal cells
• Previous studies in Dr. Guerrero-Cazares’ lab have corroborated that the mechanism by which NPC’s migrate involves Slit proteins and their corresponding receptor, known as a Roundabout or Robo receptor. These two components make up the Slit/Robo signaling pathway
• Previous studies in Dr. Guerrero-Cazares’ lab have corroborated that the Slit/Robo signaling pathway participates in modulating GBM cell migration speed:
• Previously, patient derived GBM cells were cultured and treated with shRNA to decrease the expression of the Robo1 receptor.
• Two different shRNA sequences were used to create 2 different Robo1 KD cell variants
• Additionally, an EV and SCR shRNA were used to create 2 negative control cell variants
• Here in this first graph we see the cell variant on the x-axis and migration speed on the y-axis.
• The lab found that the Robo1KD cells showcased a decreased basal level migration speed than the WT, EV, and SCR cells.
• The lab then found that the Robo1KD cells showcase a change in their cell volume
• Here in this other graph, we see the cell variants on the X-axis and the cell volume on the y-axis, we see that the Robo1KD cells showcase a significant increase in cell volume.
• From these preliminary results, we can propose that one possible way the Slit/Robo regulates migration is by modulating the cell volume regulatory genes and actin cytoskeleton dynamics.
• We hypothesize that: The Slit/Robo signaling pathway modulates the expression levels and protein localization of cell volume and actin cytoskeleton associated proteins.
• We aimed to determine the role of the Slit/Robo signaling pathway in modulating cell volume regulatory genes and actin cytoskeleton regulatory proteins.
The methods of my current study are similar to what I previously explained where
• Patient derived GBM cells were cultured and grown in media
• Two different shRNA sequences were used to create 2 different Robo1 KD cells: KD1=shRNA1 and KD2=shRNA2
• An empty vector (EV) and scrambled shRNA sequence (SCR) were used to create negative controls
• We then conducted various experiments in the presence and absence of Slit2 protein in the culture media
• Analysis was done by RT-qPCR, WB, and Immunocytochemistry
The results of Dr. Zarco’s and my experiments involves cell volume, actin cytoskeleton proteins, and the localization of those proteins.
I will first talk about the results on cell volume associated genes
Our lab has already corroborated that changes in cell volume can influence the migratory capacity of GBM cells
• But here in this study, we wanted to corroborate and analyze specifically the role of the Robo1 receptor in regulating genes that modulate cell volume in GBM cells.
• We chose the following 4 genes because they are known to have important roles in regulating the cell volume of GBM cells specifically
• Here are 4 graphs showing the RT-qPCR results of genes responsible for regulating cell volume
• In all the graphs, the X-axis indicates the cell variant and the y-axis as the relative mRNA levels of the gene of interest normalized to the WT cells.
• In this first graph, the gene of interest is AQP4 which is a water channel transporter. This water channel increases in expression in the KD cells
• In this second graph, the gene of interest is NKCC1 which is a sodium, potassium, chloride transporter. This ion transporter increases in expression in the KD cells.
• In this third graph, the gene of interest is KCC1 which is a potassium chloride cotransporter protein. This ion transporter increases in expression in the KD cells.
• In this fourth graph, the gene of interest is NCC which is a sodium chloride symporter. This ion symporter increases in expression in the KD cells.
• From these, we can conclude that these 4-cell volume regulatory genes are directly affected by the Robo1 receptor since we see a significant increase in all of them in the KD cells.
Next, we wanted to corroborate the role of the Slit/Robo signaling pathway in modulating the expression of proteins associated with the actin cytoskeleton.
• First, we see Western Blot images of various genes involved in regulating actin cytoskeleton dynamics.
• To orient you, we have 2 experimental groups: control cells or cell that were treated with the Slit2 protein.
• The horizontal top line, indicates the cell variants
• The genes of interest are on the left of the figure
• Below, we see a quantification of the western blot results from 3 independent experiments: with the x-axis indicating cell variant and the y-axis indicating the relative level of protein of interest
• The results are easier explained using this graph, but the same exact data is seen in the western blot on the above.
• For this first portion of the western blot we examined the expression of phospho-cofilin
• In cells, Cofilin promotes the regeneration of actin filaments by severing preexisting filaments.8
• When cofilin is phosphorylated on Ser3, the protein is inactivated and all cofilin-actin interactions are inhibited. 8
• In the control group which shown in the graph as the black columns, we see a significant increase in the expression of phospho-Cofilin in the KD cells
• This indicates Slit2 is possibly interacting with other Robo receptors, and that p-cofilin is regulated by these receptors as well
• In the presence of Slit, we see a decrease in the expression of p-Cofilin in all cell variants
• However, in the presence of Slit2, the KD cells cannot reach their lowest possible levels of p-cofilin seen in the WT, EV, and SCR cells
• This correlates with the less migratory phenotype
• It is also important to note that this is not due to an increase in total cofilin expression. The second panel represents total-cofilin expression which indicates that the total-cofilin protein expression remains unchanged, and it is only the p-cofilin expression that changes amongst the cell variants
• For this second portion of the western blot we examined the expression of phosphor-ERM
• Phospho-ERM is a protein that is responsible for crosslinking the actin filaments of the cell to the plasma membrane and is activated when it is phosphorylated.
• Previous literature indicates that low levels of p-ERM reduces proliferation and invasion
• total-ERM represents the total amount of phosphorylated and unphosphorylated ERM protein, which we also examined by western blot
• In the control group which shown in the graph as the black columns, we see a decrease in the expression of phospho-ERM in the KD cells
• In the presence of Slit, we see an increase in the expression of p-ERM in the WT, EV and SCR cells
• However, in the presence of slit, the KD cells do not recuperate to basal condition levels of p-ERM expression. They remain at low levels of p-ERM expression
• This means that the KD cell cannot respond to the stimulus of slit because the Robo1 receptor is absent meaning that the Slit/Robo signaling pathway is critical in modulating the expression of p-ERM
• It is also important to note that this is not due to an increase in total ERM expression. The fourth panel represents total-ERM expression which indicates that the total-ERM protein expression remains unchanged, and it is only the p-ERM expression, which is the active form of ERM, that changes amongst the cell variants
• Lastly, I would like to mention that GAPDH serves as our control across all cell lines since it is involved in the glucose metabolism pathway
• Lastly, we wanted to corroborate and analyze specifically the role of the Slit/Robo signaling pathway in modulating the localization of cytoskeleton associated proteins in GBM cells
• This is critical in understanding the mechanism by which the Slit/ Robo signaling pathway modulates GBM migration
• During migration, structures call Focal Adhesion Complexes form and help in rearranging and connecting the actin cytoskeleton to the ECM
• By immunocytochemistry, we examined 4 proteins associated with Focal adhesion complexes to better understand how this signaling pathway modulates their localization in GBM cells.
• The first protein we examined was FAK which aids in linking integrins to the actin cytoskeleton
• The second protein is Paxillin which links cell surface receptors to the actin cytoskeleton
• The third protein is Neuronal Wiskott Aldrich syndrome protein or NWASP protein which stimulates actin polymerization
• And the fourth protein is Vinculin which links integrins to the actin cytoskeleton
• These are representative images of the localization of the proteins in the SCR and Robo1 & 2 KD cells
• There are 3 rows indicating the cell variant: SCR, KD1 and KD2
• And there are 5 columns indicating which protein is fluorescing
• In this first column, the green indicates the actin cytoskeleton
• We see that the cell morphology is significantly different between the SCR cells and the KD cells
• In the second column we see FAK fluorescing in red: in the SCR cells we see a diffuse layout of FAK but the KD cells showcase a concentration in a ring structure along the outside of the cell
• This same pattern is seen in Paxillin in the next column
• In Figure B: we are examining NWASP and Vinculin
• Here, we see in the second column NWASP is fluorescing in red: here we see that in the KD cells, NWASP primarily resides around the nucleus and as we reach the outer edge of the cell we see a decrease in distribution of NWASP, unlike the SCR cell.
• We see this same pattern in the distribution of Vinculin
From these results, we can conclude that:
• The Slit/Robo signaling pathway increases the expression levels of genes that contribute to cell volume regulation in GBM cells.
• And that:
• The mechanism by which the Slit/Robo signaling pathway causes the pro-migratory phenotype is by the activation of Cofilin and ERM.
• The future directions of this study include discovering drug-specific molecules to possibly inhibit this signaling pathway
• The literature reports only one such drug known as Robo-N which inhibits Slit protein effects by acting as a decoy receptor for Robo1 and hijacking the ligand
• We hope to investigate other molecules that inhibit this signaling pathway
• Finding an inhibitory drug molecule would improve GBM treatment by decreasing the migratory capacity of GBM cells, and thus the recurrence rate
• This would increase overall patient survival
• Lastly, I would like to thank my PI Dr. Guerrero-Cazares, my mentor at the Mayo Clinic, Dr. Zarco, our funding resources, and most importantly the patients for providing invaluable resources.
• Thank you for your time and I hope you enjoyed my presentation!

Streaming Media 1

Rights Statement

http://rightsstatements.org/vocab/InC/1.0/