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 potential. 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 PIK3CA(H1047R), one of the most frequent mutations occurring in human breast cancer, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CA(H1047R) 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 PIK3CA(H1047R) on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CA(H1047R) tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

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Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

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The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

Cellular differentiation and lineage commitment in adult tissues is a tightly regulated, yet highly dynamic process. It is of crucial importance to understand how different cell fates are maintained and how cellular plasticity is regulated: In the context of normal development but also in the light of the fact that perturbations acting as cell fate switches are the underlining cause for many human diseases. Still, the regulation of cell fate in many tissues, including the breast, remains largely elusive. We performed a high-content confocal image-based loss-of-function screen for regulators of primary breast cell fate. We infected primary human breast cells with a shRNA tumor suppressor library in serum-free suspension culture in an arrayed format and assessed a variety of phenotypic parameters using automated confocal imaging. We found that removal of tumor-suppressor genes increased the number of progenitor cells and promoted self-renewal potential. Unexpectedly, these effects were particularly linked to an increase in the number of cells of the luminal lineage. Redundant shRNA activity (RSA) and pathway analysis revealed that activation of the Hippo pathway was significantly associated with this increase in self-renewal and the luminal phenotype. We focused on the Hippo core kinases LATS1/2 (LATS), which were among the highest scored hits in the initial screen. Depletion of LATS in normal breast cells derived from several independent donors promoted self-renewal, luminal fate and increased the numbers of luminal progenitors. Luminal progenitors have been shown to include the cell-of-origin of most human breast cancers and supporting this hypothesis, we found that LATS protein expression was low or absent in most human breast cancers. Furthermore, we generated a gene signature of LATS depleted primary breast cells, which revealed a high enrichment in genes belonging to luminal A and B subtypes of breast cancer. Surprisingly, endogenous activation of the Hippo pathway via removal of its core kinases LATS only resulted in modest upregulation of its canonical targets YAP/TAZ, but rather triggered a pronounced increase in the expression of luminal and luminal progenitor genes. These findings challenge the current observation that Hippo effectors YAP/TAZ are associated with mammary basal cell fate and uncover a novel YAP/TAZ-independent role of LATS in the regulation on breast cell fate. Citation Format: Adrian Britschgi, Stephan Duss, Sungeun Kim, Heike Brinkhaus, Joana Pinto Couto, Duvini De Silva, Loren Miraglia, Michael B. Stadler, Anthony P. Orth, Ghislain M.C. Bonamy, Venkateshwar A. Reddy, Mohamed Bentires-Alj. Hippo kinases LATS1/2 control human breast cell fate. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3305.