Jess McThomas is a rising Junior. She is a Health Science Major with Neuroscience, Biology, and Chemistry minors. She enjoys watching criminal minds until the depths of the early morning, then coming into lab on two hours of sleep and a large iced coffee.
Everett Gillis is a rising Junior. He is a Biochemistry and Molecular Biology Major. He loves spending time jamming to heavy metal music that he listens to while hitting his newest deadlift PR.
Jenna King is a rising Junior. She is a Health Science Major with a Chemistry Minor. She is a fun-loving person that helps keep spirits high on days that do not go very well in the lab.
Dr. Jenna Craig is the mentor you wish you had. She played Basketball and received her BS in Molecular Biology at Millersville University and received her PhD in Genetics at The Pennsylvania State University. An athlete, doctor, mother, and mentor all in one. What more could you ask for!
Some Background on Bladder Cancer
Bladder cancer (BC) is often described as a heterogeneous disease in that a bladder cancer tumor consists of cells with varying molecular subtypes within a singular tumor. Bladder cancer cells are often classified according to their gene expression profiles and to their morphologies, which are physical structures observable with microscopy. These characteristics give rise to three broad categories of BC cell lines: luminal, basal, and non-type, which are referred to as molecular subtypes.
Luminal bladder cancer is a less aggressive and invasive form of BC, whereas basal bladder cancer is comparatively more aggressive and invasive, often leading to a worse patient outcome. Luminal and basal bladder cancer have opposing gene expression profiles which may cause the 5 change in aggression and invasiveness. So, what exactly is the driving force behind the differences in gene expression between the molecular subtypes?
The goal of our lab is to answer this question using various molecular biology techniques to study gene expression changes and possible regulatory mechanisms that alter the gene expression differences between luminal and basal BC. DNA methylation, the process of reducing gene expression by binding methyl groups to DNA, is a mechanism of importance in our research. We believe that DNA methylation is a major regulatory mechanism that dictates gene expression profiles of BC tumor cells and ultimately contributes to tumor heterogeneity. Forkhead Box A1 (FOXA1) has been shown to be regulated by DNA methylation specifically in basal BC. Thus, DNA methylation of FOXA1 and likely other genes, may contribute to clonal evolution of BC cells into a basal molecular subtype. In addition, because patients have far worse outcomes with basal BC, DNA methylation is likely to contribute to more aggressive and invasive forms of this disease. We hypothesize that Retinoblastoma protein 1 (RB1) is a negative regulator of aberrant DNA methylation, thus, in luminal BC cells such as UMUC1 where RB1 is expressed, FOXA1 is over-expressed.
Genetics for Dummies- some general definitions
KO (KnockOut) cells: CRISPR-Cas9 technology was used to permanently stop the expression of RB1, a tumor suppressor gene that inhibits the transcription of genes involved with cell growth. Mutations in the RB1 gene prevent it from making functional protein, making the cell unable to regulate cell division.
WT (WildType) cells: Natural cell line without any genomic manipulation.
CpG islands: Our genes have regions of repeated cytosine-guanine (CG) base pairs called CpG islands. These regions are highly susceptible to methylation of cytosines, which is critical for controlling gene expression.
FOXA1 (Forkhead Box A1): Forkhead box A1 is a transcriptional regulator and pioneer factor, making its effects on the genome as precise as the regulation of single gene expression and as broad as the unwinding of heterochromatin
RB1 (Retinoblastoma Protein 1: Retinoblastoma protein 1 is a cell cycle inhibitor which may be lost in advanced bladder cancer. Its loss is linked to the reduced expression of other genes, such as FOXA1
A Deeper Dive into our Projects
For my project, I’m attempting to do a site-specific DNA methylation assay through means of bisulfite conversion. Our gene of interest, FOXA1, is known to have three different CpG islands along its sequence, yet only one of them is known to control its expression, CpG 143. In our knockout cell line of RB1, a decreased expression of FOXA1 was discovered, and we now hypothesize that RB1 may have a role in the methylation of CpG island 143. My job is to determine if that’s true. So far this summer, I have been growing WT and KO cells in order to isolate DNA samples. With pure DNA samples, I will be able to convert the genomic sequence by using a method called bisulfite conversion. The goal is to convert all unmethylated cytosines to uracil, and leave all methylated cytosines as-is. I will run a PCR using primers from CpG 143 to amplify the sequence and, if time allows, send them out for sequencing. If cytosines are present in the bisulfite-converted sequence, there is methylation present, and the opposite is true if there are little to no cytosines. Ideally, if cytosines are present in our bisulfite-converted WT sequence, but not present in our converted KO sequence, then RB1 may play a role in the methylation status of FOXA1!
Currently, I’m in the process of cleaning my isolated DNA samples, as I’ve been having some setbacks in finding a process that works. Luckily, we found a breakthrough method and hope to be converting our DNA soon!
CHARACTERIZATION OF RB1-DEFICIENT BC BY CDK 4/6 INHIBITOR RESPONSE AND CD274 EXPRESSION.
Bladder cancer (BC) tumor heterogeneity drives drug resistance and complicates treatment. The mechanism underlying progression of heterogeneity has been proposed the compounding effect of mutations amounting to cellular evolution. Interestingly, mutation resulting in retinoblastoma protein 1 (RB1) loss is associated with higher degree and muscle invasive bladder cancer. This loss is known to decrease cell cycle inhibition and change immune system interaction. Given a luminal BC cell line and CRISPR-edited RB1 mutant, along with two naturally occurring strains exhibiting RB1 suppression, I characterized RB1 loss by cyclin dependent kinase (CDK) 4/6 inhibitor response and PD-L1 expression.
RB1-deficient BC responds to selective cell cycle inhibitor
CDK 4/6 inhibitor Palbociclib has been demonstrated an effective immunotherapy in breast and bladder cancers. The therapeutic inhibits cyclin D-CDK4/6 complexes from deactivating RB1 and promoting cell cycle progression. Traditionally having been implemented only for the treatment of RB1-positive tumors, the drug has more recently undergone testing in RB1-deficient tumors. We explored the response of UMUC1 wild type (WT) and RB1 knockout (KO) to Palbociclib with an endpoint assay.
Our data were consistent with the hypothesis that KO samples would exhibit increased drug susceptibility (Fig. 1). This examination uniquely isolated RB1 status in CDK 4/6 inhibitor testing and underscored the promise of Palbociclib in novel applications.
Immune ligand RNA expression increased in RB1-deficient BC:The PD-1 pathway mediates body cell recognition by lymphocytes via the connection of a cell-surface PD-L1 ligand with a T-cell PD-1 receptor. The interaction neutralizes immune responses to healthy body cells but may be leveraged by cancerous cells which express PD-L1. Targeting inappropriate ligand expression, treatment with pembrolizumab monoclonal antibody therapy demonstrated successful disruption of the PD-1 pathway in studies of advanced BC. RB1 implication in a related PD-L1 pathway prompted exploration of PD-L1 expression in cases of RB1 deficiency.
Through RT-qPCR, we identified increased PD-L1 (CD274) RNA expression in RB1-knockout UMUC1 and two established RB1-deficient cell lines (Fig. 2). These exploratory experiments prompted RB1 and CD274 RNA and protein assessments between more numerous cell lines, though further tests would have been cost prohibitive. Regardless, PD-L1 modulation by RB1 speaks to the therapeutic susceptibility which mutation incurs and should be explored.
RB1 stands a consequential biomarker whose pathways offer therapeutic targets to be exploited. While mutation associates with poorer patient outcomes, it brings vulnerabilities to be leveraged in the development and application of highly selective treatments.
I performed a quantitative polymerase chain reaction (RT-qPCR) to quantify gene expression in bladder cancer cells. This allowed me to compare the relative amounts of RNA expressed between cell lines. Previously, Dr. Craig looked at the difference in FOXA1 expression between WT and RB1 KO UMUC1 with RT-qPCR and western blotting. Her findings supported the hypothesis that FOXA1 gene expression would decrease significantly with the removal of RB1, resulting in a more aggressive and invasive cell line.
My goal was to find and measure the expression of a gene of characteristics like FOXA1. I identified Fibroblast Growth Factor Receptor 3 (FGFR3) as a gene of a comparable profile. Like FOXA1, FGFR3 has CpG islands that are within the introns and exons of the gene. With this information, we hypothesized FGFR3 would also have decreased gene expression in our RB1 KO cell line compared to the WT. FGFR3 instructs the creation of various proteins which play roles in the regulation of cell growth and proliferation. In muscle invasive bladder cancers (like our UMUC1 cell line), FGFR3 mutations and decreased expression are associated with worse prognoses due to their poor reaction to chemotherapy.
I ran qPCR plates RB1 KO F and RB1 KO B cells to compare FGFR3 RNA expression to a WT control. In each plate, I used probes for 18S (a gene expressed in all cells to serve as a control), FOXA1, RB1, and FGFR3. Using FOXA1 as our standard, I analyzed the expression of FGFR3. In RB1 KO cells, FGFR3 and FOXA1 RNA were suppressed in comparison to WT cells.
These data underscored the severity of RB1 mutations in BC. RB1 mutations are associated with a poorer patient outcome and can impede patient response to treatment. Witness to FGFR3 suppression elicited by RB1 loss in vitro, we hope to improve understanding of RB1 as a biomarker to help better predict patient responses to therapeutics.