PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer.

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 PDGFRß-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells.

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529-37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869-84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

The ninth ENBDC Weggis meeting: growth and in-depth characterisation of normal and neoplastic breast cells.

Mammary gland biologists gathered for the ninth annual workshop of the European Network for Breast Development and Cancer (ENBDC) at Weggis on the shores of Lake Lucerne in March 2017. The main themes were oestrogen receptor alpha signalling, new techniques for mammary cell culture, CRISPR screening and proteogenomics.

Anticipating mechanisms of resistance to PI3K inhibition in breast cancer: a challenge in the era of precision medicine.

Frequent subversion of the PI3K (phosphoinositide 3-kinase) pathway during neoplastic transformation contributes to several hallmarks of cancer that result in a competitive advantage for cancer cells. Deregulation of this pathway can be the result of genomic alterations such as PIK3CA mutation, PTEN (phosphatase and tensin homologue deleted on chromosome 10) loss or the activation of upstream protein tyrosine kinases. Not surprisingly, the PI3K signalling pathway has become an attractive therapeutic target, and numerous inhibitors are in clinical trials. Unfortunately, current therapies for advanced cancers that target PI3K often lead to the development of resistance and relapse of the disease. It is therefore important to establish the molecular mechanisms of resistance to PI3K-targeted therapy. With the focus on breast cancer, in the present article, we summarize the different ways of targeting PI3K, review potential mechanisms of resistance to PI3K inhibition and discuss the rationale of combination treatments to reach a balance between efficacy and toxicity.

Cell-based bioluminescence screening assays.

Drug screening is an essential and widely used technique for drug discovery in various biomedical fields notably in oncology. Here we describe a functional screening assay based on the bioluminescence detection of a secreted luciferase for monitoring cell viability of cancer cells in a high-throughput format. This assay allows the screening of large libraries comprising thousands of compounds and the identification of potential anticancer molecules in a rapid, facile, and cost-effective manner.

Multiplex blood reporters for simultaneous monitoring of cellular processes.

Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted Vargula luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted Gaussia luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously.