Reduced JAG1 Expression Through miR-200 Overexpression or Crispr-Cas Mediated Knockout Impairs TNBC Growth and Metastasis

Abstract

Studies from our lab demonstrated that increasing miR-200 expression in human triple negative breast cancer (TNBC) reduced tumor growth and metastasis In Vivo. In this study, we found that overexpression of miR-200s in TNBC cells significantly reduced the expression of JAG1. When JAG1 was knocked out in MDA-MB-231 cells proliferation and invasion were significantly reduced In Vitro. Moreover, loss of JAG1 inhibited mammary tumor growth and metastasis In Vivo. RNA sequencing revealed that loss of JAG1 altered the expression of genes associated with the ECM, angiogenesis, and EMT. These results imply that miR-200s may mediate some of their antitumor actions through reducing JAG1 expression and suggest that agents targeting JAG1 should be further evaluated as a therapeutic strategy for TNBC.

Keywords: JAG1; TNBC; breast cancer; metastasis; miR‐200.

FIGURE 1

(A) Heatmap from RNA sequencing data of MDA‐231EV and MDA‐231c141 tumors showing members of the Jagged‐Notch family that were differentially expressed between the two cell lines. (B–L) qPCR data for the Jagged‐Notch genes shown in the heatmap for MDA‐231EV and MDA‐231c141 tumors. The individual values have been plotted from 5 independent tumors from each cell line with the mean and SEM shown using the horizontal line and error bars. (M) Western blot and quantification of the western blots for (N) JAG1 and (O) HES1 in MDA‐231EV and MDA‐231c141 tumors. β‐actin was used as a loading control for the western blots. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Both miR‐200 clusters were overexpressed in the human TNBC cell line MDA‐MB‐436. (A) shows that 4 out of 5 miR‐200 family members were significantly overexpressed in MDA‐436‐200f cells compared to MDA‐436EV control cells. Elevated expression of the miR‐200 family in MDA‐MB‐436 cells induced (B) a significant reduction in JAG1 mRNA and (C, D) JAG1 protein. (E) Tumor growth curves for MDA‐436EV and MDA‐436‐200f cells injected into the 4th mammary gland of NCG mice. MDA‐436‐200f cells had (F) significantly delayed tumor onset and (G) significantly reduced tumor volume at 63 days post injection. (H) qPCR confirming that JAG1 was significantly reduced in MDA‐436‐200f tumors compared to MDA‐436EV tumors. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

(A) qPCR and (B) western blot for JAG1 in control (231JAG1Con) or two different JAG1 knockout clones (231JAG1KO‐K and 231JAG1KO‐R). (C) qPCR and (D) western blot analysis for HES1 in 231JAG1Con, 231JAG1KO‐R, and 231JAG1KO‐R cells. β‐actin was used as a loading control for the western blots. Representative flow plots for (E, I) 231JAG1Con, (F, J) 231JAG1KO‐K, and (G, K) 231JAG1KO‐R cells for (E–G) BrdU and (I–K) Annexin V with the quantification of three independent trials for (H) BrdU and (L) Annexin V. (M–O) representative invasion assay images with (P) the quantification of three independent invasion assay trials. Each point on the graph represents an independent trial, and the mean and SEM are shown. *p < 0.05, ***p < 0.001.

FIGURE 4

(A) Tumor onset and (B) tumor growth curves for 231JAG1Con or 231JAG1KO cells injected into the 4th mammary gland of NCG mice. (C) Tumor growth as determined by tumor specific growth rate (SGR) was significantly reduced in 231JAG1KO tumors compared to 231JAG1Con tumors. Since all tumors were collected when they reach ~10% of the mouse's body weight (D) there was no significant difference in the size of the primary mammary tumors at the time of collection. Reduced levels of (E) JAG1 mRNA and (F) HES1 mRNA were maintained in the mammary tumors.

FIGURE 5

Hematoxylin and eosin stained images of the two independent primary mammary tumors induced by (A–D) the injection of 231JAG1Con cells at (A, C) low or (B, D) high magnification or (E–H) the injection of 231JAG1KO cells at (E, G) low or (F, H) high magnification. Error bars in A, C, E, G are 100 μm while error bars in B, D, F, H are 30 μm.

FIGURE 6

Vimentin immunohistochemistry showing tumor cells in (A–E) lungs and (F–J) livers of two independent mice bearing (A, B, F, G) 231JAG1Con tumors or (C, D, H, I) 231JAG1KO tumors. Quantification of the (E) lung and (J) liver metastatic tumor burden for mice bearing 231JAG1Con or MDA‐231JAG1KO tumors. Scale bars are 1000 μm and ****p < 0.0001.

FIGURE 7

(A) Hierarchical clustering of the gene expression profiles of 231JAG1Con and 231JAG1KO tumors using all genes, Pearson distance measure and average linkage clustering. (B) Heatmap of the top 35 differentially expressed genes in 231JAG1KO versus 231JAG1Con tumors based on the false discovery rate.

FIGURE 8

Immunohistochemistry for (A, B) NOTCH1, (C, D) NOTCH2, (E, F) NOTCH3, and (G, H) NOTCH4 in a representative (A, C, E, G) 231JAG1Con tumor or (B, D, F, H) 231JAG1KO tumor. Scale bars are 60 μm.