Understanding drivers of doxorubicin resistance in Atypical Teratoid Rhabdoid Tumor Open Access
Palkar, Antara (Spring 2023)
Abstract
Atypical teratoid rhabdoid tumor (ATRT) is a rare, aggressive pediatric central nervous system cancer primarily affecting children under 3 years of age. The median survival rate for ATRT is poor, at 9 to 17.8 months. While patients may respond initially to therapy, around 70% have recurrence and the 5-year survival from recurrence or relapse was less than 10%. We hypothesize that relapsed ATRT cells are resistant to chemotherapies such as doxorubicin and are now interested in understanding how this resistance is being conferred and sustained. Our lab found that OLFML2A was significantly overexpressed in doxorubicin resistant cancer cells generated from CLF_PEDS0005T_T1, a Renal Medullary Carcinoma (RMC) line. We hypothesized that OLFML2A may be overexpressed in other SMARCB1 deficient cancers, including ATRTs, Malignant Rhabdoid Tumor (MRT) and RMC. However, our findings suggest that OLFML2A may only play a role in MRT and RMC, and may not affect ATRT cells. Our lab also previously generated a doxorubicin resistant ATRT cell line, BT16 DR. Through whole transcriptome sequencing, we found FOXP1 and ABCB1 to be significantly upregulated in BT16 DR. We hypothesized that overexpression of these genes conferred doxorubicin resistance. BT16 cells with overexpression of FOXP1 did not lead to doxorubicin resistance. However, BT16 DR cells with overexpression of FOXP1 had a 9-fold increase in IC50 compared to controls. Additionally, we observed that with FOXP1 overexpression, BT16 cells seemed to aggregate more but did not confer a proliferative advantage. Overexpression of ABCB1 has yet to be evaluated in our cell lines; however, literature suggests ABCB1 plays an important role in modulating resistance in other cancer types. Finally, we did not find evidence of FOXP1 overexpression modulating ABCB1 expression in either parental or resistant lines. Thus, these findings suggest FOXP1 is not sufficient or required for resistance to doxorubicin in ATRT but enhances doxorubicin resistance.
Table of Contents
Introduction 1
Methods 5
Figure 1. Expression of OLFML2A, FOXP1 and ABCB1 in HA1E and HT1376 as indicated by DepMap data 7
Table 1. Cell line confluency for Cell-Titer Glo Assay 8
Table 2. List of primers used for qRT-PCR analysis 9
Table 3. List of primers used for BP reaction 11
Results 11
Figure 2. IC50 data between parental and DR cell lines 12
Figure 3. OLFML2A levels across RNA-seq, qRT-PCR and immunoblot in SMARCB1 deficient cell lines 14
Figure 4. Viral efficiency as measured by 293T expression of OLFML2A expression post transfection 15
Figure 5. RNA-seq results comparing gene expression in parental compared to doxorubicin resistant BT16s 17
Figure 6. Level of expression of FOXP1 across SMARCB1 deficient cell lines 19
Figure 7. Immunoblot detailing FOXP1 expression across nuclear and cytoplasmic fractions across the BT16 and BT16 DR cell lines 20
Figure 8. Level of expression of FOXP1 in the FOXP1 overexpressed BT16 lines 21
Figure 9. Cell counts in Luciferase and FOXP1 overexpressed cell lines 22
Figure 10. Morphological changes in the FOXP1 overexpressed cells 23
Figure 11. FOXP1 expression conferring doxorubicin resistance 24
Figure 12. Level of ABCB1 in FOXP1 overexpressed BT16 and BT16 DR cells 26
Figure 13. RNA-seq of BT16 FOXP1 vs BT16 parentals 27
Figure 14. RNA-seq of BT16 DR FOXP1 vs BT16 DR LUC 28
Figure 15. BSRGI digest of ABCB1 overexpression vectors 29
Discussion 29
References 36
About this Honors Thesis
| School | |
|---|---|
| Department | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Keyword | |
| Committee Chair / Thesis Advisor |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Understanding drivers of doxorubicin resistance in Atypical Teratoid Rhabdoid Tumor () | 2023-04-08 11:56:55 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|