Signaling Network Assessment of Mutations and Copy Number Variations Predicts Breast Cancer Subtype-specific Drug Targets

Individual cancer cells carry a bewildering number of distinct genomic alterations i.e., copy number variations and mutations, making it a challenge to uncover genomic-driven mechanisms governing tumorigenesis. Here we performed exome-sequencing on several breast cancer cell lines which represent two subtypes, luminal and basal. We integrated this sequencing data, and functional RNAi screening data (i.e., for identifying genes which are essential for cell proliferation and survival), onto a human signaling network. Two subtype-specific networks were identified, which potentially represent core-signaling mechanisms underlying tumorigenesis. Within both networks, we found that genes were differentially affected in different cell lines; i.e., in some cell lines a gene was identified through RNAi screening whereas in others it was genomically altered. Interestingly, we found that highly connected network genes could be used to correctly classify breast tumors into subtypes based on genomic alterations. Further, the networks effectively predicted subtype-specific drug targets, which were experimentally validated.Comment: 4 figs, more related papers at http://www.cancer-systemsbiology.org, appears in Cell Reports, 201

Comparison of the thymidine kinase genes from three entomopoxviruses

The entomopoxviruses (insect poxviruses) of eastern spruce budworm (Choristoneura fumiferana), two year cycle spruce budworm (C. biennis) and the Indian red army worm (Amsacta moorei) are being studied in our laboratory for their potential as biological insecticides and expression vectors. These viruses characteristically replicate in the cytoplasm of insect cells and produce occlusion bodies that serve to protect the virion from the environment. By analogy to mammalian pox-viruses, they should also contain a viral thymidine kinase (TK) that functions in viral DNA synthesis. The replication of the A. moorei entomopoxvirus was inhibited by bromodeoxyuridine whereas the baculovirus of Autographa californica was insensitive to this drug. This result was a biochemical indication that entomopoxviruses contained a kinase that phosphorylated this nucleoside analogue and thus viral DNA synthesis was inhibited. TK genes from the three different insect poxviruses were identified, cloned and sequenced. The sequences of the TK genes of the entomopoxviruses were closely related and exhibited 63.2% identity and 9.9% similarity at the protein level. However, there was only 36.7% identity and 13.6% similarity when these enzymes were compared to their mammalian poxvirus counterpart in vaccinia virus. Finally, one entomopoxvirus TK gene was expressed in Escherichia coli mutants lacking the enzyme. These bacteria were converted to a phenotype that could incorporate radioactive thymidine into their chromosomal DNA. The results presented in this paper provide impetus for the design of a recombinant entomopoxvirus expression system in which foreign genes could be introduced into the viral TK locus under selective pressure from bromodeoxyuridine.NRC publication: Ye

Differential sensitivity of A549 non-small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors

It has been demonstrated that A549 non-small cell lung cancer (NSCLC) cells are sensitive to epidermal growth factor receptor (EGFR) inhibitors in in vivo xenograft animal models, but are relatively resistant in conventional in vitro monolayer growth assays. Here, we utilized anchorage-independent cell growth/survival assays as well as motility assays and demonstrated that these tests detect the effects of two EGFR inhibitors, the small molecule inhibitor AG1478 and the ligand-blocking antibody 225 mAb, on A549 cells more sensitively than monolayer growth assays. AG1478 was more effective than 225 mAb at inhibiting EGF-stimulated anchorage-independent cell growth, in part due to its pronounced ability to inhibit cell survival, whereas 225 mAb and AG1478 were both able to inhibit cell motility. In order to determine which EGFR signalling pathway components were most strongly associated with these cell responses, we analyzed in parallel the phosphorylation levels of EGFR itself as well as several downstream pathway elements. We found that the limited ability of 225 mAb to inhibit MAPK, PI3K and STAT3 phosphorylation correlated with its inability to promote anchorage independent apoptosis, but did not correlate with its ability to inhibit motility. Based on our results in A549 cells, we propose that EGF stimulates tumour progression of NSCLC largely through effects on anchorage-independent growth and survival, as well as motility.NRC publication: Ye

Preclinical analysis of acquired resistance after prolonged efficacious metronomic oral topotecan chemotherapy plus pazopanib therapy in advanced ovarian carcinoma

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy - the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. The anti-VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.Peer reviewed: YesNRC publication: Ye

Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma

Metastatic spread of melanoma to CNS is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to undelying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (ednrb) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as bcl2a1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The pro-metastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDRNB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor, A192621, translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.Peer reviewed: YesNRC publication: Ye