Mesenchymal tumor cells drive adaptive resistance of Trp53-/- breast tumor cells to inactivated mutant Kras

Abstract

As precision medicine increases the response rate of treatment, tumors frequently bypass inhibition, and reoccur. In order for treatment to be effective long term, the mechanisms enabling treatment adaptation need to be understood. Here, we report a mouse model that, in the absence of p53 and the presence of oncogenic Kras^G12D , develops breast tumors. Upon inactivation of Kras^G12D , tumors initially regress and enter remission. Subsequently, the majority of tumors adapt to the withdrawal of Kras^G12D expression and return. Kras^G12D -independent tumor cells show a strong mesenchymal profile with active RAS-RAF-MEK-ERK (MAPK/ERK) signaling. Both Kras^G12D -dependent and Kras^G12D -independent tumors display a high level of genomic instability, and Kras^G12D -independent tumors harbor numerous amplified genes that can activate the MAPK/ERK signaling pathway. Our study identifies both epithelial-mesenchymal transition (EMT) and active MAPK/ERK signaling in tumors that adapt to oncogenic Kras^G12D withdrawal in a novel Trp53^-/- breast cancer mouse model. To achieve long-lasting responses in the clinic to RAS-fueled cancer, treatment will need to focus in parallel on obstructing tumors from adapting to oncogene inhibition.

Keywords: Kras; breast cancer; mouse model; treatment adaptation.

Fig. 1

Mammary tumor formation in the Trp53 knockout (KO) iKras^G12D mouse model (A) Schematic showing the Trp53 KO iKras^G12D mouse model and the tumor growth curve. (B) Tumor growth curves of tumors where Kras^G12D activation led to tumor growth and Kras^G12D inactivation (day 0) led to either a reactivated tumor (RT, n = 11) or led to a not reactivated tumor (nRT, n = 8). (C) Table showing the data displayed in B. Median period to doxycycline (dox) withdrawal is time between the injection of tumor cells to dox withdrawal. Median period to RT is the time between dox withdrawal and a minimum of doubling of the tumor in size after remission with continued growth thereafter.

Fig. 2

The RT loses EpCAM expression upon Kras^G12D inactivation and becomes mesenchymal‐like (A) Representative FACS plots of PT, RT, and 6 days after dox withdrawal (d6) tumors. EpCAM and CD49f expression of fluorescent tumor cells, lineage (Ter119/CD31/CD45)‐DAPI. PT n = 3, d6 n = 3, RT n = 3. (B) The percentage of EpCAM^high and EpCAM^low cells in the PT, d6, and RT tumors. PT n = 14, d6 n = 4, RT n = 10. Mean ± SEM is shown (2‐way ANOVA, **P < 0.005, ****P < 0.0001). (C) Pathology, H&E staining. PTs have an epithelial, invasive phenotype. RT have a stromal, spindle‐like, mesenchymal‐like, invasive phenotype. Scale bar is set at 100 μm.

Fig. 3

RTs display a strong (epithelial‐mesenchymal transition) EMT profile. Comparison of the transcriptomes of 4 PTs and 4 RTs. (A) Gene Set Enrichment Analysis (GSEA) hallmark gene sets upregulated in PTs and RTs (nom. P‐value < 0.05, FDR q‐value < 0.25). (B) Top 10 gene ontology terms enriched in PT. (C) Top 10 gene ontology terms enriched in RT. (D) Heat map of the expression of epithelial and mesenchymal marker genes.

Fig. 4

RTs activate RAS‐RAF‐MEK‐ERK (MAPK/ERK) signaling (A) Simplified schematic of the MAPK/ERK phosphorylation signal transduction cascade. (B) Volcano plot displaying the transcriptome highlighting differential expressed genes in color (FDR < 0.01, fold change > 1.5). MAPK Pathway Activity Score genes [42] are indicated. PT n = 4, RT n = 4. (C) Immunofluorescence staining for phosphorylated ERK1/2 (pERK1/2) (red), GFP (green), and DAPI (blue) of tissue deriving from a normal Krt14‐Cre mT/mG mammary gland, a PT, and an RT. Scale bar is set at 10 μm. Images of a biological replicate are shown in Fig S5. (D) The percentage of cells expressing pERK1/2 in PTs and RTs. Quantification of each 4 images of 2 biological controls of PTs and RTs is shown. Data are represented as mean ± SEM (unpaired t test, *P < 0.05). (E) The intensity of pERK1/2 staining in pERK1/2‐positive cells. Quantification of each 4 images of 2 biological controls of PTs and RTs are shown. Data are represented as mean ± SEM. (unpaired t test, *P < 0.05) (F) The cumulative intensity of pERK1/2 staining in the tumors. Quantification of each 4 images of 2 biological controls of PTs and RTs are shown. Data are represented as mean ± SEM. (unpaired t test, n.s.) (G) Model: constitutive active Kras^G12D results in strong MAPK/ERK signaling in a subset of the PT cells. RT cells do not reach as strong of a phosphorylation cascade per cell as in some PT cells. However, since a large percentage of individual RT cells activate MAPK/ERK signaling, the result for both tumor types as a whole is similar MAPK/ERK signaling.

Fig. 5

Tumors are genetically unstable and acquire multiple alterations that can activate MAPK/ERK signaling (A) Number of total acquired mutations by each PT and RT. The black horizontal line shows the median. (B) Venn diagrams showing the overlap and divergence of mutations acquired by a PT and its matched RTs (RT 1, 2, 3) and the overlap between mutations acquired by RTs originating from the same PT (mouse 1, mouse 2) (Venn diagram tool: [43]). (C) Copy number profiles with thresholds of ± 0.25 for gains/losses and ± 1 for amplifications/deletions are shown for PT and matched RTs compared with their matched normal genome. Heatmaps show PTs and their 3 matched RTs (RT 1, 2, 3) in both mice (mouse 1, mouse 2) separately. Red indicates gain/amplified and blue loss/deleted regions. (D) Absolute copy number profiles genome‐wide for PTs and the 3 matched RTs are shown. Absolute copy numbers are computed based on logR values derived from genomic segments using Sequenza. The profiles are shown separately for the 2 mice (mouse 1, mouse 2). (E) Oncoplot showing genes directly involved with MAPK/ERK signaling, 4 out of 11 have genetic alterations. RTs (RT 1, 2, 3) are shown directly next to their matched PTs. (F) Oncoplot showing receptor tyrosine kinase (RTK) genes with genetic alterations. RTs (RT 1, 2, 3) are shown directly next to their matched PTs.