Differences in gynecologic tumor development in Amhr2-Cre mice with KRASG12D or KRASG12V mutations

Abstract

How different KRAS variants impact tumor initiation and progression in vivo has not been thoroughly examined. We hypothesize that the ability of either KRAS^G12D or KRAS^G12V mutations to initiate tumor formation is context dependent. Amhr2-Cre mice express Cre recombinase in tissues that develop into the fallopian tubes, uterus, and ovaries. We used these mice to conditionally express either the KRAS^G12V/+ or KRAS^G12D/+ mutation. Mice with the genotype Amhr2-Cre Pten^(fl/fl) Kras^G12D/+(G12D mice) had abnormal follicle structures and developed low-grade serous ovarian carcinomas with 100% penetrance within 18 weeks. In contrast, mice with the genotype Amhr2-Cre Pten^(fl/fl) Kras^G12V/+ (G12V mice) had normal follicle structures, and about 90% of them developed uterine tumors with diverse histological features resembling those of leiomyoma and leiomyosarcoma. Granulosa cell tumors also developed in G12V mice. Differences in cell-signaling pathways in the uterine tissues of G12D and G12V mice were identified using RNA sequencing and reverse-phase protein array analyses. We found that CTNNB1, IL1A, IL1B, TNF, TGFB1, APP, and IL6 had the higher activity in G12V mice than in G12D mice. These mouse models will be useful for studying the differences in signaling pathways driven by Kras^G12V/+ or Kras^G12D/+ mutations to aid development of targeted therapies for specific KRAS mutant variants. Our leiomyoma model driven by the Kras^G12V/+ mutation will also be useful in deciphering the malignant progression from leiomyoma to leiomyosarcoma.

Figure 1

Morphologic comparison of ovaries from 9-week-old (a) Pten^fl/fl Kras^G12D/+ Amhr2-Cre (G12D) and (b) Pten^fl/fl Kras^G12V/+ Amhr2-Cre (G12V) mice. The ovaries from the G12D mice had substantial defects in follicle development, but those from the G12V mice appeared to have normal follicle development. A, antral follicle; AF, atretic antral follicle; CL, corpus; P, primordial and primary follicle; PF, preantral follicle; ANF, abnormal follicle. The box highlighted epithelial hyperplasia.

Figure 2

G12D and G12V mice had different types of gynecologic tumors. (a) Low-grade serous ovarian carcinoma with metastatic peritoneal tumor nodules developed in G12D mice at week 27. (b–d) Ovarian stromal tumors, uterine leiomyoma, and uterine sarcoma developed in G12V mice from week 40 to week 56.

Figure 3

Western blot analysis of pERK expression in uterine tissues from KRAS-mutant mice and immunohistochemical analysis of pERK expression in human leiomyoma. (a) Activation of pERK in uterine tissues from G12D and G12V mice. The mice consisted of the following: #13 (14.1 weeks old, Pten^fl/fl Amhr2-Cre, control), #10 (14.1 weeks old, G12D), #12 (14.1 weeks old, G12D), #1416 (14.4 weeks old, G12D), #38 (19.6 weeks old, G12D), #1406 (14.7 weeks old, Pten^fl/fl Amhr2-Cre, control), #1402 (14.7 weeks old, G12V), #1411 (14.7 weeks old, G12V), and #1412 (14.7 weeks old, G12V). #13, #10, and #12 were littermates, as were #1402, #1411, and #1412. (b) Upregulation of pERK during uterine tissue progression to leiomyoma in G12V mice. (c) Immunostaining for pERK in 10 human leiomyoma samples.

Figure 4

Significantly differentially expressed proteins in the uterine and ovarian tissue of G12D and G12V mice as determined using RPPA analysis. These analyses were performed using protein lysates extracted from the ovarian and uterine tissues of three G12D, three G12V, and three control mice. The values shown are the protein expression levels in the control, G12V and G12D mice. Statistical significance was determined using an unpaired t-test with the Welch correction. *p < 0.05; **p < 0.01. ns, not significant; OV, ovarian; UT, uterine. The welch’s t test of significance and figures were generated with GraphPad Prism version 8.0.0 for macOS, GraphPad Software, San Diego, California USA, www.graphpad.com.

Figure 5

Differences in the activation of canonical pathways in uterine and ovarian tissues from approximately 14-week-old G12D and G12V mice as determined using RPPA analysis. Activation and inhibition of canonical pathways were identified via Ingenuity Pathway Analysis using RPPA data on differentially expressed proteins (Supplementary Table 1) in the ovarian and uterine tissues of three G12D mice, three G12V mice, and three control mice without KRAS mutations. An absolute z-score of 2 implied activation or inhibition of the corresponding pathways. The figures were generated through the use of IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis).

Figure 6

Differences in the activity of upstream regulators of gene networks in uterine tissues from G12D and G12V mice as discovered using RNA-seq analysis. The upstream regulators of gene networks were identified via Ingenuity Pathway Analysis using the uterine tissue gene expression profiles (Supplementary Table S2 and S3) from five G12D mouse (14 to 38-week-old), five G12V mice (15 to 37-week-old), and five control mice with the Amhr2-Cre Pten^fl/fl genotype (14 to 66-week-old). The uteri from the G12D and control mice appeared to have normal phenotypes, but the G12V mice had normal phenotype and leiomyoma phenotypes. The most activated and inhibited gene network upstream regulators (absolute z-score > 5) are shown. (a) Gene/protein upstream regulators that activate or inhibit the gene networks in G12V mice in comparison with those in G12D mice. (b) Drug/chemical upstream regulators that activate or inhibit the gene networks. The figure was generated through the use of IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis).