Proliferation Genes Repressed by TGF-ß Are Downstream of Slug/Snail2 in Normal Bronchial Epithelial Progenitors and Are Deregulated in COPD.

Slug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-ß in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-ß, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-ß are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-ß,we show that there is positive correlations between the effect of differentiation and TGF-ß on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and ß-Catenin, two molecules from TGF-ßsignaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-ß-induced mechanisms.

PARP inhibitors and radiation potentiate liver cell death in vitro. Do hepatocellular carcinomas have an achilles' heel?

BACKGROUND: A promising avenue for cancer treatment is exacerbating the deregulation of the DNA repair machinery that would normally protect the genome. To address the applicability of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) combined with radiotherapy for the treatment of hepatocellular carcinoma (HCC) two approaches were used: firstly, the in vitro sensitivity to the PARPi Veliparib and Talazoparib +/- radiation exposure was determined in liver cell lines and the impact of the HBV X protein (HBx) that deregulates cellular DNA damage repair via SMC5/6 degradation was investigated. Secondly, PARP expression profiles and DNA damage levels using the surrogate marker gammaH2AX were assessed in a panel of control liver vs HCC tissues. METHODS: Cell cytotoxicity was measured by clonogenic survival or relative cell growth and the DNA damage response using immunological-based techniques in Hep3B, PLC/PRF/5, HepG2- and HepaRG-derived models. Transcriptome changes due to HBx expression vs SMC6 loss were assessed by RNA sequencing in HepaRG-derived models. PARP and PARG transcripts (qPCR) and PARP1, H2AX and gammaH2AX protein levels (RPPA) were compared in control liver vs HBV-, HCV-, alcohol- and non-alcoholic steatohepatitis-associated HCC (tumor/peritumor) tissues. RESULTS: PARPi cytotoxicity was significantly enhanced when combined with X-rays (2Gy) with Talazoparib having a greater impact than Veliparib in most in vitro models. HBx expression significantly lowered survival, probably driven by SMC5/6 loss based on the transcriptome analysis and higher DNA damage levels. PARP1 and PARP2 transcript levels were significantly higher in tumor than peritumor and control tissues. The HBV/HCV/alcohol-associated tumor tissues studied had reduced H2AX but higher gammaH2AX protein levels compared to peritumor and control tissues providing evidence of increased DNA damage during liver disease progression. CONCLUSIONS: These proof-of-concept experiments support PARPi alone or combined with radiotherapy for HCC treatment, particularly for HBV-associated tumors, that warrant further investigation.

Integrative analysis of multi-platform reverse-phase protein array data for the pharmacodynamic assessment of response to targeted therapies.

Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.

Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

Analytical Platforms 1: Use of Cultured Cells and Fluorescent Read-Out Coupled to NormaCurve Normalization in RPPA.

The analytic platform described in this chapter uses proteins extracted from cultured cells as an infinite source of material to set up, validate, and quality control an RPPA platform. Readout of the arrays uses near-infrared fluorescence labeling and data normalization is performed using the bioinformatics package NormaCurve.In the first part, we will describe the advantages, drawbacks, and different applications of cell line material for RPPA. In the second part, we will describe how the staining protocol, the method of readout, and the normalization method applied afterward are interconnected and should be considered together. Finally, we will describe the NormaCurve package, which is freely available, and its requirements for implementation.Four protocols are provided in this chapter: (1) Protein lysis of cell lines using a homemade Laemmli buffer, (2) RPPA staining for fluorescent readout including a signal amplification step, (3) total protein staining in the visible spectrum for normalization purposes, and (4) total protein staining in the near-infrared spectrum for normalization purposes.

PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma.

Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with BAP-1 (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient's tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM.

Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response.

BACKGROUND AND AIMS: Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response. METHODS: We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response. RESULTS: The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect. CONCLUSION: LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

Inhibition of mTOR downregulates expression of DNA repair proteins and is highly efficient against BRCA2-mutated breast cancer in combination to PARP inhibition.

Breast cancer is a complex disease in which each patient could present several genetic alterations that are therapeutically relevant in cancers. Here we explored the therapeutic benefit of combining PARP and mTOR inhibitors in a context of DNA repair deficiency and PI3K pathway activation. The combination of everolimus and olaparib was tested in BRCA2-mutated patient-derived xenografts (PDX) carrying alterations in the PI3K/AKT/mTOR pathway. An RPPA analysis of different signalling pathways was performed in untreated and treated xenografts. Everolimus and olaparib showed marked anti-tumor activities in the monotherapy setting and high efficacy when given in combination with 100% of mice showing tumor regressions. The fraction of P-H2AX positive cells was increased in both monotherapy arms and strongly increased in the combination setting. Everolimus given as monotherapy resulted in downregulation of different proteins involved in DNA damage repair, including FANCD2, RAD50 and SUV39H1. In the combination setting, expression of these proteins was almost completely abolished, suggesting convergence of PARP and mTOR in downregulation of DNA damage repair components. In conclusion, our results suggest that combining mTOR and DNA repair inhibition could be a successful strategy to treat a subset of breast cancer with BRCA2 mutation and alterations in the PI3K/AKT/mTOR pathway.

Correction: Protein biomarkers predictive for response to anti-EGFR treatment in RAS wild-type metastatic colorectal carcinoma.

Supplementary Table 1 and the Supplementary Figure legends were not included when this manuscript was first published. The files are now available here.

Protein biomarkers predictive for response to anti-EGFR treatment in RAS wild-type metastatic colorectal carcinoma.

BACKGROUND: Metastatic colorectal cancer (mCRC) patients with mutant KRAS or NRAS are ineligible for anti-epidermal growth factor receptor (anti-EGFR) therapy, as RAS mutations activate downstream pathways independently of EGFR and induce primary resistance. However, even among RAS wild-type (WT) patients, only a fraction responds to anti-EGFR therapy, suggesting that other mechanisms of resistance exist. We hypothesise that different (epi)genetic alterations can lead to primary anti-EGFR resistance and that the crucial end point is the activation of protein signalling pathways. METHODS: We analysed the expression and activation of proteins involved in cell signalling, using reverse phase protein arrays, on a multicentre French cohort of RAS WT mCRC treated with anti-EGFR treatment. RESULTS: We identify activated EGFR and HER3 as protein biomarkers predictive for better overall survival. Active EGFR signalling and downstream PI3K, but not MAPK, pathway activation are associated with response to anti-EGFR treatment. Left-sided mCRC displays active ErbB2/3 and Wnt pathways and a better response to anti-EGFR therapy compared to right-sided mCRC. CONCLUSIONS: We identify active EGFR and PI3K signalling as a key factor for response to anti-EGFR treatment in mCRC and highlight the importance of developing these biomarkers in clinical practice for the selection of RAS WT mCRC patients that would benefit from anti-EGFR treatment.