Targeting ribosome biogenesis as a novel therapeutic approach to overcome EMT-related chemoresistance in breast cancer
- Department of Cardiothoracic Surgery, Weill Cornell Medicine, United States
- Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, United States
- Neuberger Berman Lung Cancer Center, Weill Cornell Medicine, United States
- Systems Medicine and Bioengineering Department, Houston Methodist Cancer Center, Houston Methodist Hospital, United States
- Department of Radiology, Houston Methodist Cancer Center, Houston Methodist Hospital, United States
- Department of Pathology and Laboratory Medicine, Houston Methodist Cancer Center, Houston Methodist Hospital, United States
- Department of Cell and Developmental Biology, Weill Cornell Medicine, United States
Figures
Figure 1 with 4 supplements
Activation of ribosome biogenesis pathway during the epithelial-to-mesenchymal transition (EMT) transitioning phase in Tri-PyMT cells.
(A) Representative flow cytometry plot displays the percentage of RFP+, Double+ (Doub+, EMT transitioning cells), and GFP + Tri PyMT cells in culture. (B) Western blot of EMT markers with …
-
Figure 1—source data 1
EMT marker genes that were used in calculation of AUCell value of EMT status.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig1-data1-v1.docx
-
Figure 1—source data 2
Enriched pathways in GSEAs of EMT transitioning cells.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig1-data2-v1.docx
-
Figure 1—source data 3
Raw western blot images of Figure 1B.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig1-data3-v1.zip
-
Figure 1—source data 4
Whole western blot images of Figure 1B with labels.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig1-data4-v1.pdf
Figure 1—figure supplement 1
Characterization of fluorescent marker switch in Tri-PyMT cells.
(A) RFP + Tri PyMT cells were sorted via flow cytometry and cultured in DMEM with 10% FBS. Plot shows the percentage of RFP+, GFP + and Doub+ cells at different generations of cell culture. The …
Figure 1—figure supplement 2
RNA-sequencing analysis of Tri-PyMT cells.
RFP + Doub+, and GFP + Tri PyMT cells were purified using flow cytometry sorting, total RNA was extracted and submitted for RNA-sequencing analysis. (A) Heatmap of selected epithelial-to-mesenchymal …
Figure 1—figure supplement 3
The single-cell RNA (scRNA)-sequencing analysis of Tri-PyMT cells.
RFP+, Doub+, and GFP + Tri PyMT cells were sorted via flow cytometry and submitted for scRNA-sequencing analysis. (A) Quality controls of the scRNA-seq. Plots show the single-cell QA/QC analyses …
Figure 1—figure supplement 4
RT-PCR analysis of ribosome biogenesis (RiBi) genes in Tri-PyMT cells.
RFP+, Doub+, and GFP + cells were purified from Tri-PyMT (p7) culture using flow cytometry sorting. Total RNA was extracted and analyzed by RT-PCR. The heatmap shows the z-score of RiBi-related …
Figure 2 with 2 supplements
Activation of ribosome biogenesis (RiBi) pathway in mesenchymal-to-epithelial transition (MET) during lung metastasis outgrowth.
(A) Schematic of MET induction. GFP + Tri PyMT cells were sorted by flow cytometry (left) and injected into mice through the tail vein. Metastasis-bearing lungs were harvested after 4 weeks and …
Figure 2—figure supplement 1
The single-cell RNA (scRNA)-sequencing analysis of GFP + Tri PyMT cells.
GFP + Tri PyMT cells, including Epcam + and Epcam- cells were sorted from metastasis-bearing lungs and submitted for single-cells RNA sequencing analysis. (A) Quality controls of the scRNA-seq. …
Figure 2—figure supplement 2
The single-cell RNA sequencing (scRNA-seq) analysis with merged epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) cells.
Figure 3 with 1 supplement
MAPK and mTOR pathways mediated ribosome biogenesis activation in epithelial-to-mesenchymal transition (EMT) transitioning phase cells.
(A) Western blots show phospho-ERK, total ERK, phospho-mTORC1, total mTORC1, and p-rps6 in sorted RFP+, Doub+, and GFP + Tri PyMT cells following stimulation with 10% FBS at 0, 10, 30, and 60 min. …
-
Figure 3—source data 1
Raw western blot images of Figure 3A.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig3-data1-v1.zip
-
Figure 3—source data 2
Whole western blot images of Figure 3A with labels.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig3-data2-v1.pdf
Figure 3—figure supplement 1
Elevated RNA transcription activity in the Doub+ Tri PyMT cells.
Tri-PyMT cells (p7) including both RFP + and GFP + cells were cultured in a growth medium. EU incorporation assay was performed and detected with click chemistry using Alexa A647-azide. Labeled …
Figure 4 with 2 supplements
Ribosome biogenesis (RiBi) inhibition by Pol I inhibitor reduces the epithelial-to-mesenchymal transition (EMT)/mesenchymal-to-epithelial transition(MET) capability of tumor cells.
(A) Flow cytometry plots display the percentage of GFP + cells following BMH21 treatment. RFP+/EpCam + Tri PyMT cells were sorted and cultured in a growth medium with or without BMH21 for 5 days. …
-
Figure 4—source data 1
Raw western blot images of Figure 4B.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig4-data1-v1.zip
-
Figure 4—source data 2
Whole western blot images of Figure 4B with labels.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig4-data2-v1.pdf
Figure 4—figure supplement 1
Ribosome biogenesis (RiBi)inhibition impairs RNA transcription activity and reduces the epithelial-to-mesenchymal transition (EMT) capability of tumor cells.
(A) Fluorescence switch in Tri-PyMT cells with CX5461 treatment. Flow cytometry plots exhibit the percentage of GFP + Tri PyMT cells after treatment with the Pol I inhibitor CX5461. Biological …
Figure 4—figure supplement 2
Ribosome biogenesis (RiBi) modulating by knocking down ribosome proteins reduces the epithelial-to-mesenchymal transition (EMT)/mesenchymal-to-epithelial transition (MET) capability of tumor cells.
(A) Western blots show the knockdown expression of Rps24 and Rps28 in targeted Tri-PyMT cells. The numbers indicate the relative intensity of the band normalizing to the β-actin band of the sample. …
-
Figure 4—figure supplement 2—source data 1
Raw western blot of Figure 4—figure supplement 2A.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig4-figsupp2-data1-v1.zip
-
Figure 4—figure supplement 2—source data 2
Whole western blot of Figure 4—figure supplement 2A with labels.
- https://cdn.elifesciences.org/articles/89486/elife-89486-fig4-figsupp2-data2-v1.pdf
Figure 5 with 1 supplement
RNA Pol I inhibitor synergizes with chemo drug in vitro.
(A) Flow cytometry plots display the fluorescence switch of Tri-PyMT cells. Tri-PyMT cells (p5) were treated with cyclophosphamide (CTX, 2 μM), Pol I inhibitor (BMH21, 0.1 μM), or vehicle control …
Figure 5—figure supplement 1
Combination therapy of RNA Pol I inhibitor and chemo drugs.
Left, Bar graphs display Tri-PyMT cell viability following combination treatment with BMH21 and chemotherapeutic drugs at various concentrations. Bars represent the mean value of duplicated …
Figure 6 with 4 supplements
RNA Pol I inhibition synergizes with chemo drug in treating lung metastatic tumors.
(A–C) The same number of RFP + and GFP + Tri PyMT cells were injected in animals via tail vein. Animals were treated with cyclophosphamide (CTX) (100 mg/kg, once a week, i.p.), BMH21 (20 mg/kg, five …
Figure 6—figure supplement 1
Differential impact of Pol I inhibitor and chemo drug on epithelial and mesenchymal Tri-PyMT cells in vivo.
The same number of RFP + and GFP + Tri PyMT cells were injected in animals via the tail vein. Animals were treated with cyclophosphamide (CTX) (100 mg/kg, once a week, i.p.), BMH21 (20 mg/kg, five …
Figure 6—figure supplement 2
E-cadherin expression by LM2 cells in lung metastases.
qRT-PCR analysis of E-cadherin expression in LM2 cells isolated from lung metastases. LM2 cells in culture served as control. GAPDH served as the internal control for PCR. n=3 experiments, unpaired …
Figure 6—figure supplement 3
Correlation of ribosome biogenesis pathway and epithelial-to-mesenchymal transition (EMT) status in human breast cancer cells.
Single-nucleus cell RNA sequencing analysis of primary tumor cells from breast cancer patients (BC-A and BC-B, GEO database, GSE 198745). (A,D) The t-SNE plots show the heterogeneity in EMT statuses …
Figure 6—figure supplement 4
Survival curves of breast cancer patients with different ribosome biogenesis (RiBi) activities.
Gene expression data of METABRIC (A) and TCGA-PanCancer (B) was obtained from CBioPortal. Patients were grouped according to the average z-score of RiBi gene expression, RiBi High (>1) and RiBi Low …
Figure 7
Working model of ribosome biogenesis in EMP plasticity.
Tumor cells exhibit plasticity by undergoing EMT (epithelial-mesenchymal transition) and MET (mesenchymal-epithelial transition), contributing to the development of resistance to chemotherapies. …
