Cancer by Sfakianakis Alexandros

Κυριακή 1 Οκτωβρίου 2017

Shaping the Tumor Stroma and Sparking Immune Activation by CD40 and 4-1BB Signaling Induced by an Armed Oncolytic Virus

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma.

Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses.

Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells.

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Combined BTK and PI3K{delta} Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3K is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3K and BTK inhibitors.

Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3K inhibitor ACP-319 in vivo. We compared single-agent with combination therapy in TCL1-192 cell–injected mice, a model of aggressive CLL.

Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-B signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy.

Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814–23. ©2017 AACR.

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EGFR Downregulation after Anti-EGFR Therapy Predicts the Antitumor Effect in Colorectal Cancer

Anti-EGFR mAb is reported to induce EGFR internalization in colorectal cancer cells. However, the biological relevance of EGFR internalization with anti-EGFR mAb is unknown. Therefore, the relevance of EGFR downregulation with anti-EGFR mAb to antitumor activity in colorectal cancer cells was investigated. Quantification of EGFR on the cell surface before cetuximab treatment was assessed by flow cytometry, and its growth-inhibitory effects were measured by Trypan blue exclusion, in 10 RAS, BRAF wild-type colorectal cancer cell lines, but there was no significant correlation between EGFR number and its growth-inhibitory effect. However, a significant correlation existed between the percentage decrease in the number of EGFRs after cetuximab treatment and its growth-inhibitory effect in those cell lines. Treatment with TGFα, a ligand for EGFR, induced EGFR internalization in colorectal cancer cells, but most EGFRs subsequently recycled to the cell surface, consistent with previous studies. While cetuximab treatment induced EGFR internalization, most receptors subsequently translocated into the late endosome, leading to lysosomal degradation, as revealed by immunoblotting and double immunofluorescence. Cetuximab-sensitive colorectal cancer cells showed greater EGFR internalization, stronger cell growth inhibition, and more augmented apoptotic signals than nonsensitive cells. IHC for EGFR, performed using an EGFR pharmDx Kit (mouse anti-human EGFR mAb clone 2-18C9), in clinical specimens before and after anti-EGFR mAb therapy in 13 colorectal cancer patients showed a significant correlation between the response to anti-EGFR mAb and decreased staining after therapy.

Implications: This report clearly demonstrates that anti-EGFR mAb facilitates internalization and subsequent degradation of EGFRs in lysosomes, which is an important determinant of the efficacy of anti-EGFR mAb treatment for colorectal cancer. Mol Cancer Res; 15(10); 1445–54. ©2017 AACR.

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p120-Catenin Downregulation and PIK3CA Mutations Cooperate to Induce Invasion through MMP1 in HNSCC

Despite recent improvements in treatment for head and neck squamous cell carcinoma (HNSCC), half of all patients with a regional or advanced disease will die within 5 years from diagnosis. Therefore, identification of mechanisms driving the aggressive behavior of HNSCC is of utmost importance. Because p120-catenin (CTNND1/P120CTN) downregulation and PIK3CA mutations are commonly found in HNSCC, the objective of this study was to identify their impact on fundamental processes of metastasis, specifically, migration and invasion. Furthermore, this study aimed to identify the key effector proteins regulated by P120CTN downregulation and PIK3CA mutations. Studies using oral keratinocytes demonstrated that P120CTN downregulation and PIK3CA mutations increased migration and invasion. In addition, P120CTN downregulation and PIK3CA mutations resulted in elevated matrix metallopeptidase 1 (MMP1) levels. Inhibition of MMP1 resulted in decreased invasion, suggesting that MMP1 plays a critical role in HNSCC invasion. Moreover, analysis of HNSCC patient specimens from The Cancer Genome Atlas confirmed these findings. Tumors with low P120CTN and PI3K pathway mutations have higher levels of MMP1 compared to tumors with high P120CTN and no PI3K pathway mutations. In conclusion, this study demonstrates that P120CTN downregulation and PIK3CA mutations promote MMP1-driven invasion, providing a potential novel target for limiting metastasis in HNSCC.

Implications: Because of its role in invasion, MMP1 represents a novel, potential target for limiting metastasis in a subset of HNSCCs with P120CTN downregulation and PIK3CA mutations. Mol Cancer Res; 15(10); 1398–409. ©2017 AACR.

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A Massively Parallel Fluorescence Assay to Characterize the Effects of Synonymous Mutations on TP53 Expression

Although synonymous mutations can affect gene expression, they have generally not been considered in genomic studies that focus on mutations that increase the risk of cancer. However, mounting evidence implicates some synonymous mutations as driver mutations in cancer. Here, a massively parallel assay, based on cell sorting of a reporter containing a segment of p53 fused to GFP, was used to measure the effects of nearly all synonymous mutations in exon 6 of TP53. In this reporter context, several mutations within the exon caused strong expression changes including mutations that may cause potential gain or loss of function. Further analysis indicates that these effects are largely attributed to errors in splicing, including exon skipping, intron inclusion, and exon truncation, resulting from mutations both at exon–intron junctions and within the body of the exon. These mutations are found at extremely low frequencies in healthy populations and are enriched a few-fold in cancer genomes, suggesting that some of them may be driver mutations in TP53. This assay provides a general framework to identify previously unknown detrimental synonymous mutations in cancer genes.

Implications: Using a massively parallel assay, this study demonstrates that synonymous mutations in the TP53 gene affect protein expression, largely through their impact on splicing.

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Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma-Initiating Cell Invasiveness and Proliferation

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-25-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the β1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFβR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial–mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376–87. ©2017 AACR.

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Synergistic Activity with NOTCH Inhibition and Androgen Ablation in ERG-Positive Prostate Cancer Cells

The oncogenic activation of the ETS-related gene (ERG) due to gene fusions is present in over half of prostate cancers in Western countries. Because of its high incidence and oncogenic role, ERG and components of ERG network have emerged as potential drug targets for prostate cancer. Utilizing gene expression datasets, from matched normal and prostate tumor epithelial cells, an association of NOTCH transcription factors with ERG expression status was identified, confirming that NOTCH factors are direct transcriptional targets of ERG. Inhibition of ERG in TMPRSS2-ERG–positive VCaP cells led to decreased levels of NOTCH1 and 2 proteins and downstream transcriptional targets and partially recapitulated the phenotypes associated with ERG inhibition. Regulation of NOTCH1 and 2 genes by ERG were also noted with ectopic ERG expression in LNCaP (ERG-negative prostate cancer) and RWPE-1 (benign prostate–derived immortalized) cells. Furthermore, inhibition of NOTCH by the small-molecule -secretase inhibitor 1, GSI-1, conferred an increased sensitivity to androgen receptor (AR) inhibitors (bicalutamide and enzalutamide) or the androgen biosynthesis inhibitor (abiraterone) in VCaP cells. Combined treatment with bicalutamide and GSI-1 showed strongest inhibition of AR, ERG, NOTCH1, NOTCH2, and PSA protein levels along with decreased cell growth, cell survival, and enhanced apoptosis. Intriguingly, this effect was not observed in ERG-negative prostate cancer cells or immortalized benign/normal prostate epithelial cells. These data underscore the synergy of AR and NOTCH inhibitors in reducing the growth of ERG-positive prostate cancer cells.

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