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.

We have previously identified the natural product obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, and pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and characterize the molecular mechanism of action of OBT. We show here that OBT binds to cysteine residues with a particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. Using positron emission tomography (PET) imaging with the PET-tracer 2-[ 18 F]fluoro-2-deoxy-D-glucose (FDG), we confirm that OBT is able to penetrate the brain and functionally target brain tumors. Finally, we show that an OBT analog with improved pharmacological properties, including enhanced potency, stability, and solubility, retains the antineoplastic properties in a xenograft mouse model.

Reporters secreted into the conditioned medium in culture or into blood in vivo have shown to be useful tools for simple and non-invasive 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 applied to any field to detect multiple biological phenomena in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously.

We have previously identified the natural product Obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometrybased proteomics, functional studies in cancer cells, pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and charaterize the molecular mechanism of action of OBT. We here show that OBT and analogs, which have improved pharmacological properties, bind to cysteine residues with particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. module has been validated and successfully pursued as a target for small molecules that disrupt Nrf2 binding and/or induce the dissociation of Keap1 from CUL3 5, 6 . These efforts have identified several inhibitor classes, including cysteine-reactive natural products and synthetic compounds, that bind Keap1 and activate the Nrf2 pathway 7 .We have previously identified the quinone methide Obtusaquinone (OBT), as a potent antineoplastic agent with selectivity over normal cells for glioblastoma (GBM), and several other cancer types 8 . Within the scope of this research, we have demonstrated that OBT treatment increases the production of reactive oxygen species (ROS) and induces DNA damage, leading to apoptotic cell death. However, the molecular mode of action of OBT, its medicinal chemistry, and in vivo pharmacology have not been understood, which has impeded preclinical development.We here developed novel OBT analogs with improved pharmacological properties and show that OBT is a thiol-reactive compound that reacts reversibly with cysteine residues, and particularly binds to Keap1, leading to CRL3-mediated autoubiquitination and proteasomal degradation of Keap1 thereby activating the Nrf2 pathway. RESULTSOBT forms reversible covalent adducts with thiols.

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 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.

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.

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.