Isoform-specific ras activation and oncogene dependence during MYC- and Wnt-induced mammary tumorigenesis

Abstract

We have previously shown that c-MYC-induced mammary tumorigenesis in mice proceeds via a preferred secondary pathway involving spontaneous activating mutations in Kras2 (C. M. D'Cruz, E. J. Gunther, R. B. Boxer, J. L. Hartman, L. Sintasath, S. E. Moody, J. D. Cox, S. I. Ha, G. K. Belka, A. Golant, R. D. Cardiff, and L. A. Chodosh, Nat. Med. 7:235-239, 2001). In contrast, we now demonstrate that Wnt1-induced mammary tumorigenesis proceeds via a pathway that preferentially activates Hras1. In addition, we find that expression of oncogenic forms of Kras2 and Hras1 from their endogenous promoters has markedly different consequences for the progression of tumors to oncogene independence. Spontaneous activating Kras2 mutations occurring in either MYC- or Wnt1-induced tumors were strongly associated with oncogene-independent tumor growth following MYC or Wnt1 downregulation. In contrast, Hras1-mutant Wnt1-induced tumors consistently remained oncogene dependent. Additionally, Kras2-mutant tumors exhibited substantially higher levels of ras-GTP, phospho-Erk1/2, and phospho-Mek1/2 compared to Hras1-mutant tumors, suggesting the involvement of the ras/mitogen-activated protein kinase (MAPK) pathway in the acquisition of oncogene independence. Consistent with this, by use of carcinogen-induced ras mutations as well as knock-in mice harboring a latent activated Kras2 allele, we demonstrate that Kras2 activation strongly synergizes with both c-MYC and Wnt1 in mammary tumorigenesis and promotes the progression of tumors to oncogene independence. Together, our findings support a model for tumorigenesis in which c-MYC and Wnt1 select for the outgrowth of cells harboring mutations in specific ras isoforms and that these secondary mutations, in turn, determine the extent of ras/MAPK pathway activation and the potential for oncogene-independent growth.

FIG. 1.

MNU synergizes with Wnt1 and MYC in mammary tumorigenesis. (A) Mammary tumor-free survival curves for MTB/TOM mice (n = 7) injected intraperitoneally with 50 mg of MNU/kg of body weight at 6 weeks of age and induced with doxycycline at 7 weeks of age compared to untreated, doxycycline-induced MTB/TOM animals (n = 148). (B) Mammary tumor-free survival curves for MTB/TWNT animals (n = 11) subjected to the same MNU treatment as the animals represented in panel A compared to untreated, doxycycline-induced MTB/TWNT animals (n = 58). (C) Mammary tumor-free survival curves for MTB/TOM animals (n = 11) induced with doxycycline at 6 weeks of age and injected intraperitoneally with 50 mg/kg of MNU at 7 weeks of age compared to untreated, doxycycline-induced MTB/TOM animals (n = 148). (D) Mammary tumor-free survival curves for MTB/TWNT animals (n = 9) subjected to the same MNU treatment as the animals represented in panel C compared to untreated, doxycycline-induced MTB/TWNT animals (n = 58). Animals were palpated weekly for mammary tumors.

FIG. 2.

Wnt1-induced mammary tumors remain stably dependent upon Wnt1 for tumor maintenance and growth. (A) Representative growth curves of three mammary tumors that underwent repeated cycles of Wnt1 induction and deinduction, showing progressing to Wnt1 oncogene independence during the first, second, or third cycle of oncogene induction. (B) Chart displaying the percentages of tumors that remained dependent upon Wnt1 for tumor maintenance and growth at each cycle of deinduction. Blue bars represent the percentages of tumors that remained Wnt1 dependent in each round of deinduction. Maroon bars represent the cumulative percentages of Wnt1-dependent tumors determined for by each deinduction round.

FIG. 3.

Ras and MAPK pathway activities in Wnt1- and MYC-induced mammary tumors differ according to ras mutation status. (A) Immunoblots showing levels of ras-GTP, ras, phospho-Mek1/2, and phospho-Erk1/2 in Kras2 mutant MTB/TOM tumors compared to the levels seen with MTB/TOM tumors without detectable ras mutations. The results for mammary glands from doxycycline-induced and uninduced MTB/TRAS mice that permit expression of v-Ha-ras in the mammary gland are shown as negative and positive controls. β-Tubulin is shown as a loading control. (B) Immunoblots showing levels of ras-GTP, ras, phospho-Mek1/2, and phospho-Erk1/2 in Hras1 mutant MTB/TWNT tumors compared to the levels obtained with MTB/TWNT tumors without detectable ras mutations. Uninduced and induced mammary glands from MTB/TRAS mice were used as negative and positive controls. β-Tubulin is shown as a loading control.

FIG. 4.

Ras and MAP kinase pathway activity in Wnt1- and MYC-induced mammary tumors bearing mutations in different ras family members. (A) Immunoblots showing levels of ras-GTP, ras, phospho-Mek1/2, and phospho-Erk1/2 in Hras1-mutant MTB/TWNT tumors compared to the levels seen with Kras2-mutant MTB/TOM tumors. Uninduced and induced mammary glands from MTB/TRAS mice were used as negative and positive controls. β-Tubulin is shown as a loading control. (B) Immunoblots showing levels of ras-GTP and ras for MTB/TOM and MTB/TWNT tumors without detectable ras mutations or with Kras2 mutations and for MTB/TWNT tumors with Hras1 mutations. Uninduced and induced mammary glands from MTB/TRAS mice were used as negative and positive controls. β-Tubulin is shown as a loading control.

FIG. 5.

A latent K-ras^LA2 allele synergizes with MYC- and Wnt1-induced mammary tumorigenesis. (A) Mammary tumor-free survival curves for doxycycline-induced MTB/TOM/K-ras^LA2 mice (n = 30). Results for doxycycline-induced MTB/TOM (n = 43) and MTB/K-ras^LA2 (n = 33) littermates are shown as controls. (B) Mammary tumor-free survival curves for doxycycline-induced MTB/TWNT/K-ras^LA2 mice (n = 15). Results for doxycycline-induced MTB/TWNT (n = 19) and MTB/K-ras^LA2 (n = 33) littermates are shown as controls.

FIG. 6.

Ras and MAPK activity in tumors arising in MTB/TWNT/K-ras^LA2 mice. MTB/TWNT/K-ras^LA2 mice were induced with doxycycline until tumors reached 1 cm³. Tumors were subjected to biopsy, deinduced, and monitored weekly for regression behavior. (A) Immunoblots showing the levels of ras-GTP, phospho-Mek1/2, and phospho-Erk1/2 in biopsy samples from nine tumors from MTB/TWNT/K-ras^LA2 mice that were later demonstrated to be Wnt1 dependent following doxycycline withdrawal. An Hras1-mutant MTB/TWNT tumor, a Kras2-mutant MTB/TWNT tumor, and uninduced and induced mammary glands from MTB/TRAS mice were used as controls. β-Tubulin is shown as a loading control. (B) Immunoblots showing the levels of ras-GTP, phospho-Mek1/2, and phospho-Erk1/2 in biopsy samples from seven tumors from MTB/TWNT/K-ras^LA2 mice that were later demonstrated to be oncogene independent following doxycycline withdrawal. An Hras1-mutant MTB/TWNT tumor, a Kras2-mutant MTB/TWNT tumor, and uninduced and induced mammary glands from MTB/TRAS mice were used as controls. β-Tubulin is shown as a loading control.

FIG. 7.

Ras and MAPK activity in tumors arising in MTB/TOM/K-ras^LA2 mice. Immunoblots showing the levels of ras-GTP, phospho-Mek1/2, and phospho-Erk1/2 in biopsy samples from seven tumors from MTB/TOM/K-ras^LA2 mice that were later demonstrated to be oncogene independent following doxycycline withdrawal are presented. An MTB/TOM tumor lacking detectable ras mutations, a Kras2-mutant MTB/TOM tumor, and uninduced and induced mammary glands from MTB/TRAS mice were used as controls. β-Tubulin is shown as a loading control.