Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

PIK3CAH1047R induces multipotency and multi-lineage mammary tumours

This article has been updated


The adult mouse mammary epithelium contains self-sustained cell lineages that form the inner luminal and outer basal cell layers, with stem and progenitor cells contributing to its proliferative and regenerative potential1,2,3,4. A key issue in breast cancer biology is the effect of genomic lesions in specific mammary cell lineages on tumour heterogeneity and progression. The impact of transforming events on fate conversion in cancer cells of origin and thus their contribution to tumour heterogeneity remains largely elusive. Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of PIK3CAH1047R, one of the most frequent mutations occurring in human breast cancer5, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CAH1047R in lineage-committed basal Lgr5-positive and luminal keratin-8-positive cells of the adult mouse mammary gland evokes cell dedifferentiation into a multipotent stem-like state, suggesting this to be a mechanism involved in the formation of heterogeneous, multi-lineage mammary tumours. Moreover, we show that the tumour cell of origin influences the frequency of malignant mammary tumours. Our results define a key effect of PIK3CAH1047R on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CAH1047R tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mutant PIK3CA induces mammary cell plasticity.
Figure 2: Activation of PIK3CAH1047R leads to expression of basal- and luminal-lineage genes.
Figure 3: Expression of PIK3CAH1047R evokes multipotent stem-like cells.
Figure 4: The frequency of malignant tumour lesions is dictated by the cell of origin.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data sets generated for this study have been deposited in the Gene Expression Omnibus under accession numbers GSE59870, GSE59872 and GSE65411.

Change history

  • 02 September 2015

    The spelling of author J.P.C. was corrected.


  1. Van Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189–193 (2011)

    Article  ADS  CAS  Google Scholar 

  2. van Amerongen, R., Bowman, A. N. & Nusse, R. Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11, 387–400 (2012)

    Article  CAS  Google Scholar 

  3. de Visser, K. E. et al. Developmental stage-specific contribution of LGR5+ cells to basal and luminal epithelial lineages in the postnatal mammary gland. J. Pathol. 228, 300–309 (2012)

    Article  CAS  Google Scholar 

  4. Tao, L., van Bragt, M. P., Laudadio, E. & Li, Z. Lineage tracing of mammary epithelial cells using cell-type-specific Cre-expressing adenoviruses. Stem Cell Rep. 2, 770–779 (2014)

    Article  CAS  Google Scholar 

  5. The Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012)

  6. Hennighausen, L. & Robinson, G. W. Signaling pathways in mammary gland development. Dev. Cell 1, 467–475 (2001)

    Article  CAS  Google Scholar 

  7. Spike, B. T. et al. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. Cell Stem Cell 10, 183–197 (2012)

    Article  CAS  Google Scholar 

  8. Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006)

    Article  ADS  CAS  Google Scholar 

  9. Stingl, J. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006)

    Article  ADS  CAS  Google Scholar 

  10. Sleeman, K. E. et al. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J. Cell Biol. 176, 19–26 (2007)

    Article  CAS  Google Scholar 

  11. Prater, M. D. et al. Mammary stem cells have myoepithelial cell properties. Nature Cell Biol. 16, 942–950 (2014)

    Article  CAS  Google Scholar 

  12. Rios, A. C., Fu, N. Y., Lindeman, G. J. & Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014)

    Article  ADS  CAS  Google Scholar 

  13. Wang, D. et al. Identification of multipotent mammary stem cells by protein C receptor expression. Nature 517, 81–84 (2015)

    Article  ADS  CAS  Google Scholar 

  14. Samuels, Y. et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 304, 554 (2004)

    Article  CAS  Google Scholar 

  15. Koren, S. & Bentires-Alj, M. Mouse models of PIK3CA mutations: one mutation initiates heterogeneous mammary tumors. FEBS J. 280, 2758–2765 (2013)

    Article  CAS  Google Scholar 

  16. Meyer, D. S. et al. Luminal expression of PIK3CA mutant H1047R in the mammary gland induces heterogeneous tumors. Cancer Res. 71, 4344–4351 (2011)

    Article  CAS  Google Scholar 

  17. Liu, P. et al. Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms. Nature Med. 17, 1116–1120 (2011)

    Article  ADS  CAS  Google Scholar 

  18. Meyer, D. S. et al. Expression of PIK3CA mutant E545K in the mammary gland induces heterogeneous tumors but is less potent than mutant H1047R. Oncogenesis 2, e74 (2013)

    Article  CAS  Google Scholar 

  19. Plaks, V. et al. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Rep. 3, 70–78 (2013)

    Article  CAS  Google Scholar 

  20. Sleeman, K. E., Kendrick, H., Ashworth, A., Isacke, C. M. & Smalley, M. J. CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells. Breast Cancer Res. 8, R7 (2006)

    Article  Google Scholar 

  21. Meier-Abt, F. et al. Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium. Breast Cancer Res. 15, R36 (2013)

    Article  CAS  Google Scholar 

  22. Lim, E. et al. Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways. Breast Cancer Res. 12, R21 (2010)

    Article  Google Scholar 

  23. Lim, E. et al. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nature Med. 15, 907–913 (2009)

    Article  CAS  Google Scholar 

  24. Molyneux, G. et al. BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell 7, 403–417 (2010)

    Article  CAS  Google Scholar 

  25. Kinzel, B. et al. Functional roles of Lgr4 and Lgr5 in embryonic gut, kidney and skin development in mice. Dev. Biol. 390, 181–190 (2014)

    Article  CAS  Google Scholar 

  26. Sommer, C., Strähle, C., Köthe, U. & Hamprecht, F. A. Ilastik: Interactive Learning and Segmentation Toolkit. Proc. Eighth IEEE Int. Symp. Biomed. Imag. 230–233 (2011)

  27. Cicalese, A. et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell 138, 1083–1095 (2009)

    Article  CAS  Google Scholar 

  28. Deome, K. B., Faulkin, L. J., Jr, Bern, H. A. & Blair, P. B. Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res. 19, 515–520 (1959)

    CAS  PubMed  Google Scholar 

  29. Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009)

    Article  CAS  Google Scholar 

  30. Carvalho, B. S. & Irizarry, R. A. A framework for oligonucleotide microarray preprocessing. Bioinformatics 26, 2363–2367 (2010)

    Article  CAS  Google Scholar 

  31. NCBI Resource Coordinators. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 42, D7–D17 (2014)

  32. Gaujoux, R. & Seoighe, C. A flexible R package for nonnegative matrix factorization. BMC Bioinformatics 11, 367 (2010)

    Article  Google Scholar 

Download references


The authors thank R. Thierry for help with image processing and quantification, S. Bichet and A. Bogucki for assistance with immunohistochemical staining, S. Thiry for assistance with the microarray analysis, L. Gelman, S. Bourke and M. Kirschmann for help with microscopy, C. Blanpain, B. Roska, B. Kinzel, J. Tchorz and A. Isken for providing mouse lines, and members of the Bentires-Alj group for their feedback. O.A. and R.D.C. were supported by National Cancer Institute grant U01 CA141582. Research in the laboratory of M.B.-A. is supported by the Novartis Research Foundation, the European Research Council (ERC starting grant 243211-PTPsBDC), the Swiss Cancer League, the Swiss National Foundation, and the Krebsliga Beider Basel.

Author information

Authors and Affiliations



S.K. and M.B.-A. designed experiments, analysed the data and wrote the manuscript. L.R. contributed greatly to experimental design and data analysis. S.K. performed most of the experiments. L.R. and D.D.S. performed mammosphere cultures. L.R., D.D.S., J.P.C. and S.K. performed limiting dilution transplantations. M.B.S. and T.R. performed microarray data analysis. L.R. and J.P.C. quantified tumour immunohistochemistry. J.P.C. and A.B. isolated tumour RNA. A.B. performed immunoblotting. D.D.S. and T.E. provided technical assistance for several experiments. H.K. provided technical assistance for FACS experiments. O.A. and R.D.C. analysed histological tumour samples. All the authors discussed the data and participated in the preparation of the manuscript.

Corresponding author

Correspondence to Mohamed Bentires-Alj.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Scheme depicting mouse lines generated for lineage-tracing studies.

a, Lgr5-CreERT2(ref. 25) or K8-CreERT2(ref. 1) animals were crossed to transgenic lox-STOP-lox PIK3CAH1047R 16 and/or Tomato-reporter mice, generating Lgr5-CreERT2/Tomato, K8-CreERT2/Tomato, Lgr5-CreERT2/PIK3CAH1047R/Tomato and K8-CreERT2/PIK3CAH1047R/Tomato animals for lineage-tracing studies. Lgr5-CreERT2/Tomato and K8-CreERT2/Tomato animals were used as controls. b, Lgr5-CreERT2(ref. 25) and K8-CreERT2(ref. 1) animals were crossed to lox-STOP-lox PIK3CAH1047R(ref. 16) or PIK3CAWT(ref. 18) animals. Lgr5-CreERT2 and K8-CreERT2 animals were used as controls. Tamoxifen injection induces PIK3CAH1047R, PIK3CAWT and/or Tomato expression.

Extended Data Figure 2 Lgr5-CreERT2/Tomato and PIK3CAH1047R/Tomato labelling in the mammary nipple area.

a, Lgr5 expression in the nipple and distal area of Lgr5-CreERT2 glands (n = 3 mice). b, Tracing scheme. c, d, Representative images of mammary glands after 4 weeks tracing (n = 3 mice for each genotype). Scale bars, 2 mm, 100 μm (magnifications). e, Representative haematoxylin and eosin staining of an Lgr5-CreERT2/PIK3CAH1047R mammary gland with a tumour. Scale bar, 500 μm. LN, lymph node. f, Representative images, FACS plots and quantification of 4 days tracing (24 h after the last tamoxifen injection) (top: immunofluorescence: n = 3 mice; FACS: n = 5 technical replicates (each 1–2 pooled mice); bottom: immunofluorescence: n = 3 mice; FACS: n = 3 technical replicates (each 1 mouse)). Scale bars, 100 μm; 20 μm (magnifications). g, Representative FACS plots of 4-week tracing. h, Percentage of total Tomato-positive cells in the tracing experiments (Lgr5-CreERT2/Tomato: 4 days n = 5, 4 weeks n = 4, 8 and 13 weeks n = 3 technical replicates (each 1–2 pooled mice); Lgr5-CreERT2/PIK3CAH1047R/Tomato: 4 days n = 3, 4 weeks n = 3, 8 weeks n = 6 and 13 weeks n = 5 technical replicates (each 1–2 pooled mice)). i, Representative images of 7 days tracing (left n = 4 mice; right n = 2 mice). Scale bars, 100 μm, 50 μm (magnifications). Bar graphs show means ± s.e.m.; two-sided unpaired Student’s t-test; *P < 0.05; NS, not significant.

Extended Data Figure 3 Gating scheme for FACS experiments.

ad, Representative FACS plots of K8-CreERT2/Tomato (a), K8-CreERT2/PIK3CAH1047R/Tomato (b), Lgr5-CreERT2/PIK3CAWT (c) and Lgr5-CreERT2/PIK3CAH1047R (d) animals 4 weeks after tamoxifen injection. The gating strategy shown illustrates the elimination of doublets, dead cells (DAPIHi), and white blood cells (CD45+) and the sorting of Tomato- or GFP-positive mammary epithelial subsets (basal CD24LoSca1, luminal CD24HiSca1; luminal CD24HiSca1+).

Extended Data Figure 4 K8-CreERT2/Tomato and PIK3CAH1047R/Tomato labelling in the mammary gland.

a, Scheme depicting timeline of tracing experiments. b, Representative images and FACS quantifications of K8-CreERT2/Tomato and K8-CreERT2/PIK3CAH1047R/Tomato mammary glands 4 days after tamoxifen (24 h after the last tamoxifen injection) (top: immunofluorescence: n = 5 mice; FACS: n = 7 technical replicates (each 1–2 pooled mice); bottom: immunofluorescence n = 3 mice; FACS: n = 3 technical replicates (each 1 mouse)). Scale bars, 100 μm, 20 μm (magnifications). c, Representative FACS plots of 4-week Tomato tracing. d, Percentage of total Tomato-positive cells in mammary glands (K8-CreERT2/Tomato: 4 days n = 7, 4 and 8 weeks n = 4, 13 weeks n = 5 technical replicates (each 1–3 pooled mice); K8-creERT2/PIK3CAH1047R/Tomato: 4 days n = 3, 4 and 8 weeks n = 4 and 13 weeks n = 3 technical replicates (each 1–2 pooled mice)). Bar graphs show means ± s.e.m. *P < 0.05; two-sided unpaired Student’s t-test.

Extended Data Figure 5 Tracing of GFP-positive mammary subsets.

a, Percentage of GFP-labelled cells in K8-CreERT2/PIK3CAH1047R versus K8-CreERT2/PIK3CAWT animals 4 days after tamoxifen (24 h after the last tamoxifen injection) (n = 3 technical replicates, 2 mice per genotype). b, d, Representative FACS plots and percentages of GFP-positive cells in mammary gland subsets and total mammary epithelial cells 4 and 8–11 weeks after tamoxifen. c, e, Bar graphs showing total numbers of GFP-positive cells and numbers of GFP-positive cells in basal (CD24LoSca1) and luminal (CD24HiSca1; CD24HiSca1+) subsets of Lgr5-CreERT2/PIK3CAH1047R (c) and K8-CreERT2/PIK3CAH1047R (e) mammary epithelial cells. b, c, 4 weeks: non-induced control n = 3, control n = 9, PIK3CAWT n = 3, PIK3CAH1047R n = 9 sortings with each 1–4 pooled mice; 8–11 weeks: non-induced control n = 3, control n = 3, PIK3CAWT n = 3, PIK3CAH1047R n = 4 sortings with each 1–4 pooled mice. d, e, 4 weeks: PIK3CAWT and PIK3CAH1047R n = 3 sortings with each 1–5 pooled mice; 8–11 weeks: PIK3CAWT n = 4, PIK3CAH1047R n = 5–6 sortings with each 1–4 pooled mice. Bar graphs show means ± s.e.m.; two-sided unpaired Student’s t-test; *P < 0.05; NS, not significant.

Extended Data Figure 6 Expression of PIK3CAH1047R induces Akt phosphorylation.

Immunoblot and quantification of lysates from K8-CreERT2 control, PIK3CAWT and PIK3CAH1047R mammary glands 4 weeks after tamoxifen for p110α, pAkt, Akt, pan-keratin and Erk2 (loading control). n = 3 mice per genotype. Protein levels were normalized to pan-keratin for normalization of epithelial content. Bar graphs depict fold change over control lysate. Bar graph shows means ± s.d.; two-sided unpaired Student’s t-test; *P < 0.006; NS, not significant.

Extended Data Figure 7 Expression of basal- and luminal-lineage genes in PIK3CAH1047R subsets.

a, Expression heat maps of selected luminal and basal genes (left: Lgr5-CreERT2/PIK3CAH1047R versus control; right: K8-CreERT2/PIK3CAH1047R versus control). LM, mature luminal cells; LP, luminal progenitors; Myo, myoepithelial; SC, stem-cell enriched. b, c, Expression profiles of basal- and luminal-lineage genes in mammary subsets of Lgr5-CreERT2/PIK3CAH1047R compared with Lgr5-CreERT2 control (b) and K8-CreERT2/PIK3CAH1047R compared with K8-CreERT2 control animals (c). The qRT–PCR results are representative of 2–3 experiments of 4 pooled animals of each genotype. Bar graphs show means ± s.e.m.; two-sided unpaired Student’s t-test; *P < 0.05; NS, not significant; N.d., not detected.

Extended Data Figure 8 Luminal PIK3CAH1047R cells repopulate a mammary gland.

a, b, Number of outgrowths in cleared-fat pad transplantation of GFP-negative Lgr5-CreERT2 control and GFP-positive Lgr5-CreERT2/PIK3CAH1047R-expressing luminal subsets (CD24HiSca1) (a) and GFP-negative K8-CreERT2 control and GFP-positive K8-CreERT2/PIK3CAH1047R-expressing luminal subsets (left, CD24HiSca1; right, CD24HiSca1+) (b). Representative carmine-stained whole mounts (bottom). Scale bars, 500 μm. c, Representative immunostained sections. Scale bars, 50 μm. ac, Data from three independent experiments. d, Percentage of K14-, K8/18- and double-positive (K14/K8/18) colonies derived from Lgr5-CreERT2/PIK3CAH1047R, Lgr5-CreERT2 control (left, pooled data from n = 4 independent experiments (1–5 pooled mice)), K8-CreERT2/PIK3CAH1047R and K8-CreERT2 control subsets (right, pooled data from n = 3 independent experiments (1–5 pooled mice)). Total number of quantified colonies is shown. e, Representative images of colonies. Arrowheads indicate K8/18- (white), K14- (yellow) and double-positive (blue) colonies. Scale bars, 500 μm. f, Number of colonies derived from basal and luminal cells from Lgr5- and K8-CreERT2/PIK3CAH1047R and control mice. Left, pooled data from three independent sortings (each 1–5 pooled animals), total n = 8 (control), n = 10 (mutant) technical replicates for basal subset, n = 9 (control), n = 5 (mutant) technical replicates for luminal CD24HiSca1 subset and n = 8 (control), n = 4 (mutant) technical replicates for luminal CD24HiSca1+ subset. Right, pooled data from two independent sortings (each 1–5 pooled animals), total n = 8 (control), n = 10 (mutant) technical replicates for basal subset, n = 5 (control), n = 10 (mutant) technical replicates for luminal CD24HiSca1 subset and n = 6 (control), n = 9 (mutant) technical replicates for luminal CD24HiSca1+ subset. Five-hundred cells were seeded for each replicate. A colony was defined as a cell cluster of >5 cells. Bar graphs show means ± s.e.m.; two-sided unpaired Student’s t-test; *P < 0.05. g, Bar graphs showing number of spheres derived from FVB-control and PIK3CAH1047R-expressing luminal (CD24HiSca1−/+) mammary cells over three passages. Representative data (three replicates, n = 4 mice per genotype) from two independent experiments. Bar graphs show means ± s.d. *P < 0.02, two-sided unpaired Student’s t-test. h, Representative images of spheres derived from CD24HiSca1 cells in passage one (P1) and three (P3). Scale bars, 100 μm. N.d., not determined; NS, not significant.

Extended Data Figure 9 PIK3CAH1047R-evoked tumours express basal and luminal markers.

a, Representative FACS plots of Lgr5-CreERT2/PIK3CAH1047R and K8-CreERT2/PIK3CAH1047R tumours (n = 3). b, Percentages of total GFP-positive cells and GFP-positive basal (CD24LoSca1) and luminal (CD24HiSca1−/+) subsets of Lgr5-CreERT2 and K8-CreERT2/PIK3CAH1047R tumours (n = 3). Bar graphs show means ± s.e.m. NS, not significant; two-sided unpaired Student’s t-test c, Immunostaining for basal and luminal markers on serial sections of a multi-nodular rosette-type adenomyoepithelioma (Lgr5-CreERT2/PIK3CAH1047R) and adenomyoepithelioma (K8-CreERT2/PIK3CAH1047R). Scale bars, 100 μm, 50 μm (magnifications). d, Quantification of basal- and luminal-lineage markers of Lgr5-CreERT2 and K8-CreERT2/PIK3CAH1047R tumours. Each dot represents one tumour (top: K8/18, K14 and SMA n = 15, K5 n = 14, ER n = 10, PR n = 9, p63 n = 8; bottom: K8/18, K14, SMA and K5 n = 15, ER, PR and p63 n = 10). All Lgr5-CreERT2/PIK3CAH1047R tumours and 8/10 and 6/10 of K8-CreERT2/PIK3CAH1047R tumours show more than 1% of ER- and/or PR-positive cells, respectively. Bar graphs show means ± s.d. e, Representative haematoxylin and eosin stainings of tumour phenotypes. Scale bars, 100 μm. f, Percentage of benign and malignant mammary tumours.

Source data

Extended Data Figure 10 Expression profiling of K8- and Lgr5-CreERT2/PIK3CAH1047R mammary tumours.

a, Principle component analysis and dendogram of a hierarchical clustering of gene expression profiles from 10 K8- and 10 Lgr5-CreERT2/PIK3CAH1047R tumours and 2–3 reference mammary glands. Each dot indicates one sample. Circles represent K8-CreERT2 and squares represent Lgr5-CreERT2 animals expressing PIK3CAH1047R (filled symbols) or not (open symbols). b, Heat map of the top 1,000 genes that vary between K8-CreERT2/PIK3CAH1047R and Lgr5-CreERT2/PIK3CAH1047R tumours and The Cancer Genome Atlas (TCGA) human breast cancer gene signatures. Lum, luminal.

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Koren, S., Reavie, L., Couto, J. et al. PIK3CAH1047R induces multipotency and multi-lineage mammary tumours. Nature 525, 114–118 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing