Mammary Precancerous Stem and Non-Stem Cells Evolve into Cancers of Distinct Subtypes

Abstract

There are distinct cell subpopulations in normal epithelial tissue, including stem cells, progenitor cells, and more differentiated cells, all of which have been extensively studied for their susceptibility to tumorigenesis. However, normal cells usually have to progress through a precancerous lesion state before becoming a full-blown tumor. Precancerous early lesions are heterogeneous, and the cell subset that is the primary source of the eventual tumor remains largely unknown. By using mouse models that are tailored to address this question, we identified a keratin 6a-expressing precancerous stem cell (PcSC) subset and a more differentiated whey acidic protein-positive (WAP+) cell subset in mammary precancerous lesions initiated by the Wnt1 oncogene. Both cell subsets rapidly progressed to cancer upon introduction of constitutively active versions of either HRAS or BRAF. However, the resulting tumors were dramatically different in protein profiles and histopathology: keratin 6a+ precancerous cells gave rise to adenocarcinoma, whereas WAP+ cells yielded metaplastic carcinoma with severe squamous differentiation and more robust activation of MEK/ERK signaling. Therefore, both stem and non-stem cells in mammary precancerous lesions can contribute to the eventual cancers, but their differentiation status determines the resulting cancer phenotype. This work identifies a previously unknown player in cancer heterogeneity and suggests that cancer prevention should target precancerous cells broadly and not be limited to PcSC. SIGNIFICANCE: This work uses a novel mouse mammary gland cancer model to show that tumors initiated from different precancerous mammary epithelial cells are distinct.

Figure 1.. Krt6a-positive cells in K6a-tva/MMTV-Wnt1 mammary glands are enriched for precancerous stem cells.

(A) Experimental design of limiting dilution transplantation (LDT). (B) Representative whole mount image of the outgrowth of Lin-TVA+ cells in the #4 cleared fat pad of a recipient mouse. The inset shows the endogenous #2 gland of the same recipient mouse. Scale bar = 1mm. (C) Lin-TVA+ cells give rise to multiple cell lineages. IHC images show the expression of different cell type markers in the outgrowth of Lin-TVA+ cells. Scale bar = 20μm. Lin: non-epithelial cell lineage markers.

Figure 2.. Mutated HRAS, BRAF and ERBB2 can transform Krt6a+ PcSC cells in the hyperplastic lesion of MMTV-Wnt1 mammary glands.

Tumor-free survival Kaplan-Meier plots of K6a-tva/MMTV-Wnt1 mice with mammary glands intraductally infected by RCAS-HrasQ61L (A), RCAS-BRAFV600E (B), and RCAS-caErbb2 (C), respectively. RCAS-Actb-infected mammary glands were used as negative controls. n = mouse number. The gel picture below A shows that Tumors induced by RCAS-HrasQ61L contain RCAS-HrasQ61L provirus. Genomic DNA extracted from tumors from K6a-tva/MMTV-Wnt1 mice infected by RCAS-HrasQ61L was detected for RCAS-HrasQ61L provirus by PCR. The RCAS-HrasQ61L plasmid was used as positive control (+). The genomic DNA from spontaneous MMTV-Wnt1 tumor was used as negative control (−).

Figure 3.. Mutated HRAS and BRAF can transform non-PcSC WAP+ cells in the hyperplastic lesion of MMTV-Wnt1 mammary glands.

(A) Diagram of experimental design to examine whether TVA+ cells from WAP-tva/MMTV-Wnt1 contain Krt6+ cells. Co-IF, co-immunofluorescent staining. (B) Co-IF of GFP and KRT6 on mammary glands generated from experiment shown in A. The quantification of the percentages of KRT6- and KRT6A+ cells among GFP+ cells are shown next to the image. n = 3, mouse age = 13 weeks. Scale bar = 20μm. (C and D) Identification of the dosages of RCAS virus used to infect similar small numbers of cells in Krt6a-tva/MMTV-Wnt1 vs. WAP-tva/MMTV-Wnt1 mammary glands. The doses of 4×10⁶ and 1×10⁵ IUs of RVAS-GFP per gland for Krt6a-tva/MMTV-Wnt1 and WAP-tva/MMTV-Wnt1 mice, respectively, infected similar small numbers of cells (C) and percentages of cells (D). (E and F) Tumors can be induced from both KRT6A+ and WAP+ cells in MMTV-Wnt1 mammary precancerous lesions by HRASQ61L or BRAFV600E. Kaplan-Meier tumor-free survival plots of K6a-tva/MMTV-Wnt1 and WAP-tva/MMTV-Wnt1 mice infected by RCAS-HrasQ61L (D) and RCAS-BRAFV600E (E), respectively. The virus dosages used were the same as used in C and D.

Figure 4.. MEK/ERK signaling is elevated in tumors originated from WAP+ cells.

(A) Heat map of differential levels of total and phosphoproteins between tumors induced from WAP-tva/MMTV-Wnt1 mice (6 by RCAS-HrasQ61L and 7 by RCAS-BRAFV600E) and K6a-tva/MMTV-Wnt1 mice (6 by RCAS-HrasQ61L and 5 by RCAS-BRAFV600E) as detected by RPPA. Proteins elevated in tumors originated from WAP+ cells are listed on the right. (B and C) Western blot of total and phosphorylated MEK1/2 (B) and ERK (C) in RCAS-HrasQ61L-induced tumors from WAP-tva/MMTV-Wnt1 and K6a-tva/MMTV-Wnt1 mice. The quantification of Western blot is shown below each corresponding image.

Figure 5.. Tumors induced from Krt6a+ and WAP+ cells by HrasQ61L are different.

(A) Tumors originated from Krt6+ and WAP+ cells are significantly different. Representative HE staining of tumors induced from Krt6+ (left) and WAP+ (right) precancerous cells by the viruses indicated. (B) Immunohistochemical staining of the indicated markers was performed for tumors induced from K6a-tva/MMTV-Wnt1 (left) and WAP-tva/MMTV-Wnt1 (right) mammary glands by RCAS-HrasQ61L. Scale bar = 100μm. (C) Diagram to show that the cellular origin of cancer in mammary precancerous lesions defines breast cancer subtype.