Aim 1. Assess the effects of SHP2 signalling on the epigenetic programs of tumor-initiating cells
Downstream of various active receptor tyrosine kinases, Src-homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed protein-tyrosine phosphatase (PTP), transduces mitogenic, survival, cell-fate and/or migratory signals. SHP2 is fundamental to the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) pathway. Small hairpin knockdown of SHP2 decreases breast tumor growth and progression. Notably, a potent orally active allosteric inhibitor of SHP2 has been reported recently, and SHP2 inhibition is currently tested in a phase I trial focusing on advanced solid tumors. Yet, the effect of pharmacological inhibition of SHP2 on breast cancer metastases, overall survival, and epigenetic programs remains ill-defined. We thus decided to test this inhibitor in our studies using 2D and 3D in vitro models as well as different in vivo treatment settings. SHP2 pharmacological inhibition decreased ERK signaling, primary tumor growth and metastasis (Amante et al., JMGBN 2022 and manuscript in review).
We have also tested the effects of SHP2 inhibition on the global transcriptome in a several preclinical models of breast cancer. Analysis of the data shows a cell type specific transcriptional output upon SHP2 inhibition, suggesting that the initially identified “SHP2 signature” cannot be generalized to other cell types. We are currently assessing the minimal signature that can be found cross a variety of breast cancer models.
Aim 2: Address the effects of PI3K pathway hyperactivation on the epigenetic programs underpinning cell plasticity:
Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of mutant PI3K to induce multipotency during tumorigenesis in the mammary gland. We have shown that expression of PIK3CAH1047R mutant 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 tumors (please see image and Koren et al., Nature 2015).
We have generated new MCF10A cells expressing wild-type or PIK3CAH1047R. We have also generated K8-creERT2 animals with inducible PIK3CAH1047R or PIK3CAWT mice, and further crossed the progeny with Tomato-reporter mice to generate K8-creERT2/Tomato, K8-creERT2/PIK3CAWT/Tomato and K8-creERT2/PIK3CAH1047R/Tomato. We have induced transgene expression using tamoxifen and isolated K8-positive luminal cells and the newly formed basal cells from the K8 models by FACS of mammary epithelial cells. We are currently analyzing the MCF10A and the cells isolated from mice.
By analyzing transcriptomes from publically available datasets and our own data, we found that a metabolic enzyme that promotes the tumor initiating cell (TIC) state in models of aggressive breast cancer. It is upregulated in breast tumourspheres and metastases; its expression correlates with stem cell markers and with the worse clinical course. Mechanistically, loss of this enzyme increases deposition of epigenetic marks at the promoters of stem cell genes (Couto et al., manuscript in revision).
Aim 3. Delineate the effects of epigenetic regulators on normal mammary cell self-renewal and metastasis:
We have performed four independent screens: a) using a pooled shRNA library targeting epigenetic regulators; b) using an shRNAs targeting all tumor suppressors in the human genome; c) using a pooled shRNA library targeting metabolic/epigenetic regulators; and d) using a transposon mutagenesis system. We have identified potential regulators of mammary stem/progenitor cells that we are validating (a/c/d). We have used a high content confocal image-based shRNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells (b). We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2, 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 (Britschgi et al., Nature 2017).
Analysis of RNAseq from primary tumors and metastasis revealed increased glucocorticoid receptor activity in distant metastases. The glucocorticoid receptor mediates the effects of stress hormones, and of synthetic derivatives of these hormones that are used widely in the clinic as anti-inflammatory and immunosuppressive agents. We show that the increase in stress hormones during breast cancer progression results in the activation of the glucocorticoid receptor at distant metastatic sites, increased colonization and reduced survival (Obradovic et al., Nature 2019).
