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Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity

Abstract

Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes1,2. PIK3CA and P53 (also known as TP53) are the two most frequently mutated genes and are associated with different types of human breast cancers3. The cellular origin and the mechanisms leading to PIK3CA-induced tumour heterogeneity remain unknown. Here we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and the impact of mutations in this gene on tumour heterogeneity. Surprisingly, oncogenic Pik3caH1047R mutant expression at physiological levels4 in basal cells using keratin (K)5-CreERT2 mice induced the formation of luminal oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive tumours, while its expression in luminal cells using K8-CReERT2 mice gave rise to luminal ER+PR+ tumours or basal-like ERPR tumours. Concomitant deletion of p53 and expression of Pik3caH1047R accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3caH1047R in unipotent basal cells gave rise to luminal-like cells, while its expression in unipotent luminal cells gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that underwent cell fate transition upon Pik3caH1047R expression in unipotent progenitors demonstrated a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures characteristic of the different cell fate switches that occur upon Pik3caH1047R expression in basal and luminal cells, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3caH1047R activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation.

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Figure 1: Oncogenic Pik3ca expression in BCs or LCs leads to distinct tumour phenotypes.
Figure 2: Oncogenic Pik3ca expression and p53 deletion in BCs or LCs leads more frequently to highly invasive mammary tumours.
Figure 3: Oncogenic Pik3ca expression induces multipotency in unipotent luminal and basal progenitors.
Figure 4: Molecular characterization of oncogenic Pik3ca-induced multipotency.

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Gene Expression Omnibus

Data deposits

Microarrays have been deposited in the Gene Expression Omnibus under accession number GSE69290.

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Acknowledgements

C.B. is an investigator of WELBIO, A.V.K. is Chercheur Qualifié of the FNRS, C.S. is Maître de Recherche of the FNRS, M.Y.L. is supported by the Agency for Science, Technology and Research (A*STAR, Singapore) fellowship, M.O. and A.W. are supported by FNRS fellowships, R.R.G. is supported by a TELEVIE fellowship and S.B. is supported by the foundation “Amis de l’institut Jules Bordet”. The Center for Microscopy and Molecular Imaging is supported by the European Regional Development Fund and Wallonia. W.A.P. is supported by project grants from the National Health and Medical Research Council of Australia. This work was supported by the FNRS, TELEVIE, a research grant from the Fondation Contre le Cancer, the ULB fondation, the Fond Yvonne Boël, the Fond Gaston Ithier, the foundation Bettencourt Schueller, the foundation Baillet Latour, and the European Research Council.

Author information

Authors and Affiliations

Authors

Contributions

C.B. and A.V.K. designed the experiments and performed data analysis. A.V.K., M.Y.L. and M.O. performed all the experiments. S.B. and C.S. performed the bioinformatic analysis of gene expression and comparison with human breast cancer expression and gene amplification on human samples. S.R. and I.S. helped to perform the histological classification of mouse mammary tumours with regard to their similarities with human breast cancers. G.B. provided technical support. C.D. provided technical support for cell sorting. A.W. and R.R.G. helped with some experiments. W.A.P. provided animals and critically reviewed the manuscript. C.B. and A.V.K. wrote the manuscript.

Corresponding authors

Correspondence to Alexandra Van Keymeulen or Cédric Blanpain.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Tamoxifen administration has no long-term effect on the mammary gland.

a, b, Effect of TAM on mammary epithelial postnatal growth. a, b, Representative whole-mount preparations of carmine alum-stained mammary epithelium from the fourth mammary gland, showing that TAM induces a delay in mammary epithelium growth at early time points, but no difference is observed 8 weeks after TAM induction (a) and mean distance from lymph node distal edge to the distal epithelial edge 1 week, 5 weeks and 8 weeks after TAM injection or oil injection (b) (n = 6, 6, 4, 3, 5, 4 mice respectively for 1 week control (ctr), 1 week TAM, 5 weeks control, 5 weeks TAM, 8 weeks control, 8 weeks TAM). P value derived from two-sided Student’s t-test is 0.161, 0.035, 0.748 when comparing control and TAM conditions at 1 week, 5 weeks and 8 weeks, respectively. c, Percentage of YFP+ cells in LCs (CD29Lo/CD24+) and in BCs (CD29Hi/CD24+) analysed by FACS 48 h after TAM administration in K5-CreERT2/Pik3caH1047R/Rosa26-YFP and K8-CreERT2/Pik3caH1047R/Rosa26-YFP, or 1 week after doxycycline administration to K14-rtTA/TetO-Cre/Pik3caH1047R/p53fl/+ /Rosa26-YFP mice (n = 5, 6, 3 mice respectively for K5-CreERT2, K8-CreERT2 and K14-rtTA/TetO-Cre). Circles, individual data points. Scale bars, 100 µm. Error bars, s.e.m.

Extended Data Figure 2 Characterization of tumours derived from basal or luminal cells upon oncogenic Pik3ca expression.

ae, Characterization of adenomyoepithelioma (adenomyo) tumours derived from K5-CreERT2/Pik3caH1047R/Rosa26-YFP mice. fy, Characterization of tumours derived from K8-CreERT2/Pik3caH1047R/Rosa26-YFP mice. fj, Characterization of adenomyoepithelioma. ko, Characterization of myoepithelial carcinoma (C). pt, Characterization of invasive carcinoma of no special type (NST C). uy, Characterization of metaplastic carcinoma. a, f, k, p, u, Haematoxylin and eosin staining. b, g, l, q, v, p63 immunohistochemistry. c, h, m, r, w, Immunofluorescence of ER/K8. d, i, n, s, x, Immunofluorescence of K8/K14. e, j, o, t, y, Mean percentage of Ki67+ cells within tumours (n = 6, 3, 3, 1, 3 tumours and total number of cells counted = 10,408, 10,758, 11,174, 4,622, 5,732 in e, j, o, t, y, respectively) . Error bars, s.e.m. Scale bars, 10 µm.

Extended Data Figure 3 Similarities between mouse Pik3ca-derived mammary tumours and human breast cancers.

ad, Human breast tumour histologically classified as adenomyoepithelioma resembling K5-CreERT2/Pik3caH1047R/Rosa26-YFP-derived tumours (K5PIK TU). a, Haematoxylin and eosin staining. bd, p63 (b), K8/K18 (c) and K14 (d) immunohistochemistry in the human adenomyoepithelioma. eh, Human breast tumour histologically classified as metaplastic carcinoma resembling K8-CreERT2/Pik3caH1047R/Rosa26-YFP derived tumours (K8PIK TU). e, Haematoxylin and eosin staining. fh, p63 (f), K8/K18 (g) and K14 (h) immunohistochemistry in the human metaplastic carcinoma. ik, Principal component analysis (PCA) of the METABRIC patients together with murine tumours according to the expression values of the PAM50 genes common to mice and humans. i, PCA of three K5-CreERT2/Pik3caH1047R tumours (black dots) showing that these tumours cluster with human luminal B cancer subtype. j, PCA of seven K8-CreERT2/Pik3caH1047R/Rosa26-YFP-derived tumours (numbered black dots). Histological classification of each numbered tumour is described below the figure. k, PCA of two K8-CreERT2/Pik3caH1047R/p53fl/fl /Rosa26-YFP-derived tumours (K8PIKp53 TU) (black dots) showing that these tumours cluster together with human HER2+ subtype. l, Clustering of the murine tumours among human tumours of the METABRIC data set. Clustering was performed by grouping tumours presenting similar expression patterns of PAM50 genes. Colours on top of the heatmap represent the PAM50 subtypes attributed to the human tumours. The discrepancy between PCA and clustering analysis are due to the influence of HER2 low expression in these tumours, for which around 60% of PC2 relies on ERBB2 expression. Scale bars, 10 µm.

Extended Data Figure 4 Gating strategy to analyse and isolate tumour cells, LCs, and BCs according to their YFP, CD29 and CD24 profile.

ae, Dot plot FACS analysis of unicellular suspension of mammary tumour cells (in this example from K8-CreERT2/Pik3ca-H1047R/Rosa26-YFP tumour) stained for Lin (CD31, CD45, CD140a). Debris were eliminated from all events in P1 (a), doublets were discarded in P2 (b), the living cells were gated in P3 by DAPI dye exclusion (c), the non-epithelial Lin+ cells were discarded in P4 (d), and the YFP+ cells were gated in P5 (e). f. Gating strategy used for FACS analysis and cell sorting, showing the proportion of parent and total cells for each gate. Tumour cells were isolated based on their Lin profile for YFP tumours (P4 gate), or were isolated based on their YFP profile (P5 gate) for the YFP+ tumours, as described in Methods. gm, Dot plot FACS analysis of unicellular suspension of mammary cells (in this example from K5-CreERT2/Pik3caH1047R/Rosa26-YFP mice 12 months after TAM induction) stained for CD24, CD29 and Lin (CD31, CD45, CD140a). Debris were eliminated from all events in P1 (g), doublets were discarded in P2 (h), the living cells were gated in P3 by DAPI dye exclusion (i), the non-epithelial Lin+ cells were discarded in P4 (j), and the YFP+ cells were gated in P5 (k). l, m, CD29 and CD24 expression were used to gate the CD29LoCD24+ population, corresponding to LCs, and to gate the CD29HiCD24+ population, corresponding to BCs, either in YFP+ cells (l) or in Lin cells (m). n, Gating tree showing the gating strategy used for FACS analysis and sorting, showing the proportion of parent and total cells for each gate.

Extended Data Figure 5 Characterization of tumours derived from BCs or LCs upon concomitant expression of oncogenic Pik3ca and deletion of p53.

ao, Characterization of tumours derived from K14-rtTA/TetO-Cre/Pik3caH1047R/p53fl/+ /Rosa26-YFP mice. ae, Characterization of adenomyoepithelioma (adenomyo). fj, Characterization of myoepithelial carcinoma. ko, Characterization of metaplastic carcinoma. pa′, Characterization of tumours derived from K8-CreERT2/Pik3caH1047R/p53fl/fl /Rosa26-YFP mice. pt, Characterization of myoepithelial carcinoma (C). ua′, Characterization of metaplastic carcinoma. a, f, k, p, u, Haematoxylin and eosin staining. b, g, l, q, v, p63 immunohistochemistry. c, h, m, r, w, Immunofluorescence of K8/ER. d, i, n, s, x, Immunofluorescence of K8/K14. e, j, o, t, y, Mean percentage of Ki67+ cells within tumours (n = 4, 3, 3, 4, 6 tumours and total cells counted = 11,903, 10,670, 6,992, 14,743, 8,172 in e, j, o, t, y, respectively). z, Immunofluorescence of K8/HER2. a′, Immunofluorescence of E-cadherin/vimentin. Error bars, s.e.m. Scale bars, 10 µm.

Extended Data Figure 6 Oncogenic Pik3ca expression induces multipotency in unipotent luminal progenitors.

ad, Immunofluorescence showing the expression of K8/YFP (a, c) or K5/YFP (b, d) 1 week (a, b) and 7 months (c, d) after TAM injection in control K8-CreERT2/Rosa26-YFP mammary gland. e, Percentage of YFP+ cells in LCs (CD29Lo/CD24+) and in BCs (CD29Hi/CD24+) at different time points after TAM administration to K8-CreERT2/Rosa26-YFP mice (n = 3 mice per time point) showing that no YFP+ cells expressing CD29Hi/CD24+ were detected in control K8-CreERT2/Rosa26-YFP mammary glands at any time point. fh. Immunofluorescence of K14/YFP (f), p63/YFP (g), SMA/YFP (h) 8 weeks (f, g) or 10 weeks (h) after TAM administration to K8-CreERT2/Pik3caH1047/Rosa26-YFP mice, shows that the BCs arising from LCs upon oncogenic Pik3ca targeting expressed these classical markers of BCs. im, Induction of Pik3caH1047R expression in LCs in adult mice. il, Immunofluorescence showing the expression of K8/YFP (i, k) or K5/YFP (j, l) 1 week (i, j) and 8 weeks (k, l) after TAM injection in K8-CreERT2/Pik3caH1047R/Rosa26-YFP mice induced in adulthood. m, Percentage of YFP+ cells in LCs (CD29Lo/CD24+) and in BCs (CD29Hi/CD24+) at different time points after TAM administration to K8-CreERT2/Pik3caH1047R/Rosa26-YFP mice induced in adulthood (n = 4 mice per time point). n, o, Immunofluorescence of K5/YFP showing the clonal YFP expression in a single isolated LC 1 week after TAM injection (n), and 8 weeks after TAM injection showing a clone that gave rise to an LC and a BC (o) in K8-CreERT2/Pik3caH1047R/Rosa26-YFP mammary gland. Arrow in n points to the isolated LC, while arrow in o points to the newly arisen BC. p, Distribution of clones 1 week or 10 weeks after TAM injection in K8-CreERT2/Pik3caH1047R/Rosa26-YFP at clonal dose. Clones were scored as composed of only luminal cells (luminal clones), composed of only basal cells (basal clones) or composed of luminal and basal cells (mixed clones) (n = 4 mice per time point). See Methods for more details. qt, Immunofluorescence of K5/K8 (q), K5 (r), K8 (s) and K5/K8/YFP (t) shows that in wild-type mammary gland, K5 and K8 are not co-expressed (q), while K5/K8 double-positive cells are observed in K8-CreERT2/Pik3caH1047R/Rosa26-YFP mammary gland 8 weeks after oncogenic Pik3ca expression in LCs (rt). Arrows in rt point to K5+K8+YFP+ cells. u, v, RT–PCR analysis of luminal (u) or basal (v) genes in YFP+ LCs and BCs sorted from K8-CreERT2/Pik3caH1047R/Rosa26-YFP mice induced for 1 week, 4 weeks or 8 weeks, in YFP+ LCs derived from K8-CreERT2/Rosa26-YFP and in YFP+ BCs derived from K5-CreERT2/Rosa26-YFP mice induced for 8 weeks. Data for luminal genes are compared to adult wild-type LCs (u) while data for basal genes are compared to adult wild-type BCs (v) (n = 4 biologically independent samples per condition). Circles, individual data points. Scale bars, 10 µm. Error bars, s.e.m.

Extended Data Figure 7 Oncogenic Pik3ca expression induces multipotency in unipotent basal progenitors.

ac, Immunofluorescence showing the expression of K5/YFP (a, b) or K8/YFP (c) at 1 week (a) and 7 months (b, c) in control K5-CreERT2/Rosa26-YFP mammary gland. d, Percentage of YFP+ cells in LCs (CD29Lo/CD24+) and in BCs (CD29Hi/CD24+) at different time points after TAM administration to K5-CreERT2/Rosa26-YFP (n = 5, 4, 4, 3 mice for 1 week, 8 weeks, 7 months and 12 months, respectively) showing that no YFP+ cells expressing CD29Lo/CD24+ were detected in control K5-CreERT2/Rosa26-YFP mammary glands at any time point. eh, Immunofluorescence of K19/YFP (e), ER/YFP (f), PR/YFP (g), claudin 3/YFP (h), 8 months after TAM administration to K5-CreERT2/Pik3caH1047/Rosa26-YFP mice, shows that LCs arising from BCs upon oncogenic Pik3ca targeting expressed these classical markers of LCs. i, Immunofluorescence of K5/YFP showing the YFP expression in a single isolated BC 1 week after TAM injection at a clonal dose. Arrow points to the isolated BC. j, Distribution of clones 1 week or 7 months after TAM injection in K5-CreERT2/Pik3caH1047R/Rosa26-YFP at a clonal dose. Clones were scored as composed of only luminal cells (luminal clones), composed of only basal cells (basal clones) or composed of luminal and basal cells (mixed clones) (n = 3, 4 mice for 1 week and 7 months, respectively). See Methods for more details. k, l, RT–PCR analysis of luminal (k) or basal (l) genes in YFP+ LCs and BCs sorted from K5-CreERT2/Pik3caH1047R/Rosa26-YFP mice induced for 10–12 months, in YFP+ LCs derived from K8-CreERT2/Rosa26-YFP mice and in YFP+ BCs derived from K5-CreERT2/Rosa26-YFP mice induced for 10–12 months. Data for luminal genes are compared to adult wild-type LCs (k) while data for basal genes are compared to adult wild-type BCs (l) (n = 4 biologically independent samples per condition). m, Confocal microscopy analysis of immunofluorescence of YFP, Ntrk2 and K8 of mammary glands 7 months after Pik3ca expression in BCs, showing that the newly formed LCs after Pik3ca expression in BCs co-expressed Nrtk2 and K8. Arrow points to formed K8+/Nrtk2+/YFP+ cell. Circles, individual data points. Scale bars, 10 µm. Error bars, s.e.m.

Extended Data Figure 8 Molecular characterization of oncogenic Pik3ca induced multipotency.

a, b, Venn diagram representing the common and distinct upregulated (a) and downregulated (b) genes in BCs and LCs after Pik3ca expression in BCs and LCs compared to age-matched control BCs and LCs, respectively, with the name of the list of genes and number of genes in each section. The list of genes in each Venn section is provided in Supplementary Tables 2 and 3. cl, Venn diagrams representing the common genes upregulated (c, e, g, i, k) or downregulated (d, f, h, j, l) in the newly generated LCs or BCs after Pik3caH1047R expression in unipotent progenitors (c, d); in LCs and in BCs after Pik3caH1047R expression in LCs (genes regulated following the initial targeting of Pik3caH1047R in LCs, and thus reflecting the LC of origin) (e, f); in LCs and in BCs after Pik3caH1047R expression in BCs (genes regulated by Pik3caH1047R in BCs, and thus reflecting the BC of origin) (g, h); in BCs after Pik3caH1047R expression in LCs and in BCs (genes regulated by Pik3caH1047R expression in BCs, irrespective of cell of origin) (i, j); in LCs after Pik3caH1047R expression in BCs and in LCs (genes regulated by Pik3caH1047R expression in LCs, irrespective of cell of origin) (k, l). Diameter of the diagram is proportional to the number of genes it contains. The reported hypergeometric P values correspond to the probability of observing an intersection of this size by chance only, knowing the number of genes tested on a microarray chip.

Extended Data Figure 9 Genes of luminal-to-basal multipotency signature correlate with patient outcome in untreated breast cancer patients.

ad, Disease-free survival in untreated patients according to the level of expression (low = blue, intermediate = green or high = red) of the genes in the luminal-to-basal multipotency signature, namely NGF (a), INHBA (b), ITGB6 (c) and WNT10A (d), showing that genes of luminal-to-basal multipotency signature predict disease-free survival in untreated breast cancer patients. Patients expressing high levels of this signature are more prone to tumour relapse while those expressing lower levels of this signature show lower rates of relapse. The log-rank P values account for the significance of this difference.

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Van Keymeulen, A., Lee, M., Ousset, M. et al. Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity. Nature 525, 119–123 (2015). https://doi.org/10.1038/nature14665

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