Cancer Gene Therapy (2012) 19, 38–48 r 2012 Nature America, Inc. All rights reserved 0929-1903/12 www.nature.com/cgt
ORIGINAL ARTICLE Molecular network pathways and functional analysis of tumor signatures associated with development of resistance to viral gene therapy T-J Song1,2,*, D Haddad1,3,*, P Adusumilli1, T Kim1, B Stiles1, M Hezel1, ND Socci1,4,MGo¨nen5 and Y Fong1 1Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 2Department of Surgery, New York College of Medicine, Korea University, Seoul, South Korea; 3Institute for Biochemistry and Virchow Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany; 4Department of Bioinformatics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA and 5Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Replication-competent attenuated herpes simplex viruses have proven effective in killing many cancer cell lines. However, determinants of resistance to oncolytic therapy are mostly unknown. We developed viral therapy-resistant cells and examined changes in gene-expression pattern compared with therapy-sensitive parental cells. Colon cancer cell line HT29 and hepatoma cell line PLC5 were exposed to increasing concentrations of virus G207. Therapy-resistant cells were isolated and grown in vitro. Tumorigenicity was confirmed by ability of cell lines to form tumors in mice. Human Genome U133A complementary DNA microarray chips were used to determine gene-expression patterns, which were analyzed in the context of molecular network interactions, pathways and gene ontology. In parental cell lines, 90–100% of cells were killed by day 7 at 1.0 multiplicity of infection. In resistant cell lines, cytotoxicity assay confirmed 200- to 400-fold resistance. Microarray analysis confirmed changes in gene expressions associated with resistance: cell surface proteins affecting viral attachment and entry, cellular proteins affecting nucleotide pools and proteins altering apoptotic pathways. These changes would decrease viral infection and replication. Our study identifies gene-expression signatures associated with resistance to oncolytic viral therapy. These data provide potential targets to overcome resistance, and suggest that molecular assays may be useful in selecting patients for trial with this novel treatment. Cancer Gene Therapy (2012) 19, 38–48; doi:10.1038/cgt.2011.64; published online 21 October 2011 Keywords: herpes simplex virus; oncolytic viral therapy; G207; molecular networks; signaling pathways
Introduction expression analysis using complementary DNA (cDNA) GeneChip microarray Human Genome U133A Genetically engineered, attenuated herpes simplex virus (Affymetrix, Santa Clara, CA) to determine the possible (HSV) is a promising new modality to treat cancer. Such predictive gene changes in viral-resistant cells compared HSV viruses have been shown to be effective against with viral-sensitive cells. These changes may enable many cancer types, including colorectal, hepatoma, lung, identification of cancers resistant to viral therapy, thus prostate, bladder and breast.1–11 Among these viruses, avoiding undesirable side effects associated with higher G207 and NV1020 are currently in clinical trials. The doses of viral treatment. Moreover, if gene-expression efficacy of HSV therapy depends on both infectivity and changes associated with resistance to therapy are identi- replication. Although initial higher cell kill is achieved fied, new strategies can potentially be developed in order with a higher concentration of virus, this in turn has an to overcome them. increased potential for toxicity in vivo and may therefore not be desirable. In this study, we conducted gene-
Materials and methods Correspondence: Dr Y Fong, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY Cell lines and cell culture 10065, USA. One colon cancer (HT29) and one hepatoma cell line E-mail: [email protected] (PLC5) were used for constructing resistance to oncolytic *These authors contributed equally to this work. HSV. Colon cancer cell line HT29 (HTB-38) was grown in Received 22 June 2011; accepted 16 August 2011; published online American Type Culture Collection (ATCC) medium 21 October 2011 (Manassas, VA; McCoy’s 5a medium with 1.5 mmol l–1 Gene expression in therapy-resistant cancer T-J Song et al 39 L-glutamine and 10% fetal calf serum), and maintained were noted to be resistant up to an MOI of 400 via under standard cell culture conditions. cytotoxic assay. These resistant cells were then frozen and The human hepatoma cell line PLC5 (PLC/PRF/5) was used for subsequent experiments, as described below. This grown in ATCC medium (Eagle’s minimum essential resulted in a pressure selection of viral-resistant tumor –1 medium with 2 mmol l L-glutamine and Earle’s balanced cells with a differing cellular makeup than the parent cells. salt solution adjusted to contain 1.5 g l–1 sodium bicarbo- Cytotoxicity profiles were assessed by cellular LDH nate, 0.1 mmol l–1 nonessential amino acids and release assay. 1.0 mmol l–1 sodium pyruvate with 10% fetal calf serum) under standard cell culture conditions. Tumorigenicity assay To confirm the tumor origin of the resistant cell lines, 7 Virus normal parent cells (1 Â 10 ) were injected subcutaneously in the left flank of athymic nude mice, whereas resistant G207 is an engineered HSV type 1 (HSV-1) replication- 7 competent, second-generation oncolytic HSV. Con- cells (1 Â 10 ) were injected in the right flank, to assess for structed from R3616, it has a mutation of the ICP6 gene, tumor growth over a period of 14 days. deletion of both copies of the g134.5 genes and an insertion of the Escherichia coli lac Z marker gene into Preparation of RNA for microarray the U 39 gene.11,12 The g 34.5 gene deletion decreases Total mRNA preparation was performed in a 10-cm L 1 7 neurovirulence of the virus,11–13 and disruption of the (diameter) petri dish culture vessel (monolayer, 1 Â 10 cells UL39 gene eliminates ribonucleotide reductase activity, in confluence). Lysis was performed directly in the vessel thereby increasing specificity of the virus to proliferating following RNeasy mini kit protocol (Qiagen, Valencia, cells such as tumor cells.12,14–16 The G207 virus was CA). The mRNA samples were measured by spectro- propagated as previously described, and titer levels were photometer for proof of purity and hybridized to Human determined by standard viral plaque assay.17 Genome U133A cDNA microarray chips (Affymetrix) by the genomic core laboratory at Memorial Sloan-Kettering Cancer Center. Two samples each of parent and G207- Lactate dehydrogenase cytotoxicity assay resistant tumor cells were prepared and used. CytoTox 96 nonradioactive cytotoxic assay kit (Promega, Madison, WI) was used for lactate dehydrogenase (LDH) cytotoxicity assays. This is a colorimetric assay, where the Data analysis The chip images were scanned and processed to CEL intensity of color formation post addition of reagent is files using the standard GeneChip Operating Software proportional to the number of lysed cells (which release (GCOS) analysis suite (Affymetrix). The CEL files were LDH), thus allowing quantification of the proportion of then normalized and processed to signal intensities using cells killed by infection with the virus. the gcRMA algorithm from the Bioconductor library Assays were performed for both nonresistant parent for the R statistical programming system (Institute for cells and resistant cells. 1 Â 104 cells per well were plated Statistics and Mathematics, Wein, Austria; http://cran. in 24-well plates in 1 ml medium per well. After r-project.org/). All subsequent analyses were done on the incubation for 6 h, cells were infected with variable log (base 2) transformed data. To find differentially combinations of multiplicity of infection (MOI), which expressed genes, a moderated t-test was used as imple- describes the proportion of viral particles per cell. Viral mented in the Bioconductor LIMMA package (Fred toxicity was measured at certain time points over 7 days. Hutchinson Cancer Research Center, Seattle, WA, USA; Infected cells were washed with phosphate-buffered saline http://www.bioconductor.org). To control for multiple and lysed by 1.5% Triton X-100 (Dow, Midland, MI) to testing, the false discovery rate (FDR) method was used, release intracellular LDH. A total of 50 ml of the lysates with a cutoff of 0.05. Genes were then grouped into was then placed in 96-well reading plates, and after specific ontology functional groups using the Ingenuity addition of 50 ml of assay reagent-buffer solution, the Pathways Analysis software (Ingenuity Systems, Red- visible wavelength absorbance (490 nm) data were col- wood City, CA), also known as IPA. Network and lected using a 96-well plate reader. All samples were pathway analysis was then performed to find direct and analyzed in triplicate and results expressed as the indirect interactions on common same-directional up- or surviving fraction of cells, as determined by the measured down-regulated genes 42-fold. Functional analysis was absorbance of each sample relative to control untreated performed, also utilizing IPA. cell lysates.
Isolation of virus-resistant cells The two hepatoma and two colon cancer cell lines were Results cultured and serially infected with increasing G207 doses Generation of viral-resistant cells at variable MOIs ranging from 0.001 to 200 plaque- Serial passaging with increasing MOIs of G207 in the one forming units. When B80% cell kill was achieved with hepatoma and one colon cancer cell line resulted in the each dose, the surviving cells were re-cultured in fresh development of virally resistant tumor cells with differing medium, allowed to proliferate and subsequently infected cellular makeup compared with the parent cell lines. with a higher MOI of virus. This was repeated until cells These were used for our study.
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 40 Tumorigenicity the total common genes) and 38 downregulated (51.35%). To determine whether the final resistant cells were of real Genes changed by oncolytic HSV-resistant cell lines tumor origin and not the result of, for example, fibroblast in comparison with normal parent cells are listed in contamination, and to avoid other cell-related bias, Tables 1 and 2. Finally, hierarchical clustering charts tumorigenicity was confirmed via in vivo studies. Normal illustrate the similarity among the commonly changed parent cells were injected subcutaneously into the left genes (Figure 2). flank of nude mice, whereas resistant cells were injected into the right flank. All mice grew tumors in both flanks, Biologic functional analysis of common genes confirming the tumorigenicity of both G207-resistant With the help of IPA, the 74 common genes were tumor cells and parent cell samples. analyzed and grouped by cellular location and gene category. However, only 52 of the 74 genes were deemed Viral cytotoxicity function-, pathway- and list-eligible by IPA, and these Cytotoxicity of parent and resistant cells in the two cell were subsequently sorted into molecular and cellular lines was confirmed by LDH assay with repeated G207 functions, diseases and disorders, and physiologic system infection in increasing MOIs (Figure 1). Both HT29 and development and functions for analysis. PLC5 showed nearly 100% cell kill by day 7 at an MOI of 1 (Figures 1a and b). Cellular location. Of the 74 genes categorized, the Both cell lines generated for resistance showed less cell majority of commonly changed genes were usually kill than parent cell lines. Even at an MOI of 50, HT29 expressed in the cytoplasm (31 genes, 41.89%). Other demonstrated only 9.40% cytotoxicity by day 7 locations included the extracellular space (5 genes, (Figure 1c), and PLC5 showed cell growth despite viral 6.76%), plasma membrane (16 genes, 21.62%) and infection at 101.52% compared with control (Figure 1d). nucleus (11 genes, 14.86%). Eleven genes (14.86%) were of unknown location. Identification of same directional gene-fold changes common to both resistant cell lines Gene category. Gene categories included enzymes From a total of 26 318 genes identified to have changed (17 genes, 22.97%), transporters (7 genes, 9.46%), between parent and resistant cell lines, 13 181 genes transcription regulators (4 genes, 5.41%), kinases (50.08%) were upregulated and 13 137 (49.92%) were (3 genes, 4.05%), peptidases (3 genes, 4.05%), phospha- downregulated for both cell lines. In all, 575 genes were tases (3 genes, 4.05%), ligand-dependent nuclear recep- upregulated 42-fold (FDR o0.05) in the HT29 cell line tors (2 genes, 2.70%) and transmembrane receptors (4.36% of total upregulated genes) compared with 436 in (1 gene, 2%). Thirty-four genes were uncategorized PLC5 (3.31%). In contrast, 741 downregulated genes 42- (45.95%). fold (FDR o0.05) were identified in the HT29 cell line (5.64% of total downregulated genes) and 316 in PLC5 Molecular and cellular functions. The top 5 significant (2.41%). A total of 74 common genes (3.71% of a total of molecular and cellular function groups (according to 1994 genes upregulated 42-fold for both cell lines at P-value) with which common genes were involved FDR 0.05) were found to have the same directional entailed roles in cell morphology (11 genes, P ¼ 7.06E– change in both cell lines, with 36 upregulated (48.65% of 05), cellular development (11 genes, P ¼ 2.34E–04), cellular movement (13 genes, P ¼ 6.09E–04), cellular growth and proliferation (16 genes, P ¼ 6.37E–04) and cell-to-cell signaling and interaction (9 genes, P ¼ 6.49E–04).
Diseases and disorders. It is interesting to note that although there is much overlap between the diseases and disorders that common genes are known to be involved in, including those that are not directly relevant to this study, cancer was the most statistically significant disease to which the common genes were linked (30 genes, P ¼ 1.03E–04). Specific cancer-related functions included tumorigenesis, transformation, growth, migration, detachment, attachment, binding, invasion and survival of cancer cells, the functions highly relevant to our study.
Physiologic system development and functions. Similar Figure 1 Cytotoxicity with HSV-1 G207. G207-induced cell kill was to findings in the analysis of diseases and disorders, many assessed with cytotoxic LDH assay for parent and resistant cells. genes have overlapping roles in physiologic system Left column shows normal parent cells: (a) HT29, (b) PLC5. Right development and functions. Of note is that functions column reflects G207-resistant cells: (c) HT29, (d) PLC5. involving cell-mediated immune responses were second
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 41 Table 1 Genes commonly up- and down-regulated in both HSV-1-resistant cancer cell lines
Probe ID Name Symbol Fold change Average FDR
HT29 upregulated 217875_s_at Transmembrane, prostate androgen induced RNA TMEPAI 80.34 0.00734 201288_at Rho GDP dissociation inhibitor (GDI) b ARHGDIB 79.8 0.00647 205805_s_at Receptor tyrosine kinase-like orphan receptor 1 ROR1 35.09 0.01128 202437_s_at Cytochrome P450, family 1, subfamily B, polypeptide 1 CYP1B1 31.98 0.02043 212472_at Microtubule associated monoxygenase, calponin and LIM domain MICAL2 28.73 0.01073 containing 2
HT29 downregulated 212190_at Serpin peptidase inhibitor, clade E (nexin, plasminogen activator SERPINE2 À239.79 0.00818 inhibitor type 1), member 2 213135_at T-cell lymphoma invasion and metastasis 1 TIAM1 À174.4 0.00524 204971_at Cystatin A (stefin A) CSTA À90.95 0.00244 201427_s_at Selenoprotein P, plasma, 1 SEPP1 À66.94 0.00117 201061_s_at Stomatin STOM À62.98 0.0084
PLC5 upregulated 217875_s_at Transmembrane, prostate androgen induced RNA TMEPAI 223.29 0.00734 211548_s_at Hydroxyprostaglandin dehydrogenase 15-(NAD) HPGD 66.65 0.00014 203913_s_at Hydroxyprostaglandin dehydrogenase 15-(NAD) HPGD 66.53 0.00007 211549_s_at Hydroxyprostaglandin dehydrogenase 15-(NAD) HPGD 54.09 0.0056 203914_x_at Hydroxyprostaglandin dehydrogenase 15-(NAD) HPGD 48.45 0.00428 212473_s_at Microtubule associated monoxygenase, calponin and LIM domain MICAL2 47.84 0.00277 containing 2 212328_at LIM and calponin homology domains 1 LIMCH1 38.57 0.01678 212325_at LIM and calponin homology domains 1 LIMCH1 37.56 0.01102 212543_at Absent in melanoma 1 AIM1 36.23 0.0041
PLC5 downregulated 209301_at Carbonic anhydrase II CA2 À132.04 0.01044 217564_s_at Carbamoyl-phosphate synthetase 1, mitochondrial CPS1 À108.28 0.01036 201427_s_at Selenoprotein P, plasma, 1 SEPP1 À97.38 0.00117 204920_at Carbamoyl-phosphate synthetase 1, mitochondrial CPS1 À93.06 0.01736 212190_at Serpin peptidase inhibitor, clade E (nexin, plasminogen activator SERPINE2 À52.73 0.00818 inhibitor type 1), member 2 203962_s_at Nebulette NEBL À27.26 0.03241 Abbreviations: FDR, false discovery rate; HSV-1, herpes simplex virus type 1. The top five genes in each group are shown. among the top five of the list (eight genes, P ¼ 6.49E–04). were then algorithmically generated based on their Specifically, immune responses affecting costimulation, connectivity (Ingenuity Systems, 2009). quantity, selection of immune cells and aggregation seem Using IPA, 6 major networks were identified involving to be involved. the 74 genes commonly up- and down-regulated between the two cell lines, with 58 genes being deemed network Network and pathway analysis eligible (Table 1). The IPA software system enables systemic analysis of The top network in our analysis included genes with microarray and other data in a biologic context. It has a functions related to cancer, cell morphology, and cellular large knowledge base with integrated information from compromise (21 genes; Figure 4a). Network 2 genes were systems biology, including protein, gene, protein complex, involved in organismal functions, hepatic system devel- cell, cellular component, tissue, organ, small molecule and opment and function and carbohydrate metabolism (14 disease interrelationships, as well as millions of pathway genes). Cellular development and cellular growth and interactions extracted from up-to-date literature. It also proliferation were the top functions in network 3 (12 features a comprehensive ontology with over 600 000 genes). Network 4 involved functions similar to those of concepts. the above networks, including cancer, cellular movement and cellular growth and proliferation (11 genes). Network Networks analysis. Our commonly up- or down-regu- 5 functions also included those known to be involved in lated genes were overlaid onto a global molecular network cancer, energy production and small molecule biochem- developed from information contained in the Ingenuity istry (1 gene). Finally, the top functions in network 6 Pathways Knowledge Base. Networks of these focus genes involved free radical scavenging (1 gene).
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 42 Table 2 Network analysis
Focus Network Molecules in network molecules Top functions
1 Akt, ARHGDIB, CD59, CD82, CYP1B1, DUSP6, DUSP9, 21 Cancer, cell morphology, cellular compromise EFNA5, ENC1, FLNB, FN1, FSH, IL1, IL4R, INSR, Insulin, Jnk, LIMCH1, Mapk, MIRN124, MKP1/2/3/4, NR3C1, P38 MAPK, Pkc(s), PLCB1, Rac, RNA polymerase II, SLC2A1, SMAD3, STOM, Tgf-b, TIAM1, TPM1, TUBA4A, Tubulin 2 ABCC2, ANKZF1, C6ORF108, CA2, chenodeoxycholic acid, 14 Organismal functions, hepatic system CLIP2, CRH, D-glucose, DBT, dehydroepiandrosterone sulfate, development and function, carbohydrate DUSP6, DYNLL1, EHD1, EHD3, EPHA2, FAHD2A, HIF1A, metabolism HNF4A, LAD1, LAMA3, LAMB3, leukotriene C4, MIRN134 (includes EG:406924), MTUS1, NEBL, PDZK1, PRSS23, PTPRG, PXR ligand-PXR-Retinoic acid-RXRa, SLC16A4, SLC2A1, TAPBP, taurolithocholic acid, TNF, TRAPPC4
3 ATP, BCKDHA, BCKDHB, b-estradiol, COL4A5, CRH, CSTA, 12 Cellular development, cellular growth and CTSH, CTSL1, CYP2J2, DECR1, EPHA4, EPHB1, EPOR, F8, proliferation, hematological system GALNAC4S-6ST, heparin, HHEX, HPGD, JUN, LYN, development and function magnesium, magnesium-adenosine triphosphate, MAPK1, MAPK8IP1, MICAL2, NTRK1, PACSIN3, PHYH, prostaglandin F2a, SEPP1, SERPINE2, SOCS2, TGFB3, UCN 4 AGT, AIM1 (includes EG:202), ASAP2, BCL3, BMP1, CD46, 11 Cancer, cellular movement, cellular growth COL4A6, COL6A1, COL7A1, CPS1, CTNNB1, DAB1, EPHB2, and proliferation ERBB2, ETV4, FA2H, FSTL3, GRIN2B, H1F0, HTRA1, LAMC2, LTBP2, NTRK1, PMEPA1, PTN, RB1, RICS, ROR1, SH3BGRL3, SLC1A1, SRC, TGFB1, TLE4, TNS1, UBQLN4
5 MIRN224 (includes EG:407009), SYNGR1 1 Nervous system development and function, cancer, genetic disorder 6 Malate dehydrogenase (oxaloacetate-decarboxylating) 1 Energy production, small molecule (NADP), ME3 biochemistry, free radical scavenging Top six networks with associated genes and functions are shown.
Signaling and metabolic pathway analysis. Canonical and acute-phase response signaling (4.36E–02). In addi- pathways analysis identified the pathways (from the IPA tion, three metabolic pathways were statistically signifi- library of canonical pathways) that were most significant cant (Po0.05): nitrogen metabolism (P ¼ 1.56E–02); to our common gene data set. The significance of the pyruvate metabolism (P ¼ 3.38E–02); and arginine and association between the data set and the canonical proline metabolism (P ¼ 3.81E–02). Other pathways pathway was measured in two ways: (1) a ratio of the worthy of mention, although their involvement did not number of genes from the data set that map to the reach statistical significance, include G1/S checkpoint pathway, divided by the total number of genes that map regulation, p53 signaling, PTEN (phosphatase and tensin to the canonical pathway, is displayed; and (2) Fisher’s homolog) signaling and the nuclear factor-kB (NF-kB) exact test was used to calculate a P-value determining the pathway. probability that the association between the genes in the data set and the canonical pathway is explained by chance alone (Ingenuity Systems, 2009). Discussion IPA identified 52 genes eligible for pathways analysis. Top canonical pathways included pregnane X receptor Wild-type HSV is a broad-spectrum human virus known (PXR)/PXR activation (P ¼ 3.14E–03), nitrogen metabo- to infect a wide range of cell types. This is likely because lism (P ¼ 3.14E–03), hepatic fibrosis/stellate cell activa- of multiple cell surface receptors, including glycoprotein tion (P ¼ 3.14E–03), pyruvate metabolism (P ¼ 3.14E–03) D and nectin-1.18–20 Conditionally replicating viruses and peroxisome proliferator activated receptor-a have gained increasing attention for their ability to kill (PPARa)/retinoid X receptor-a (RXRa) activation tumor cells by oncolysis and apoptosis, and this (P ¼ 3.14E–03) (Figure 3). More specifically, six signaling attenuated strain has shown promise as a selective pathways were statistically significant: PXR/PXR act- anticancer agent.18 It has been reported that previous ivation (P ¼ 3.14E–03); hepatic fibrosis/stellate cell exposure to wild-type HSV-1 does not abrogate subse- activation (P ¼ 3.14E–03); PPARa/RXRa activation quent in vivo cancer treatment using G207 mutant (P ¼ 3.14E–03); interleukin-4 (IL-4) signaling (3.49E–02; HSV-1.21 However, from our laboratory studies on Figure 4b); caveolar-mediated endocytosis (4.26E–02); various cancer cell lines, serial challenges of stepped
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 43
Figure 2 Hierarchical clustering chart/heat map. Heat map looking at differential changes of common genes. For this case each gene is normalized as a mean expression of zero. Blue shows samples with expression below mean and red is expression above mean. increasing doses of G207 in vitro may either isolate and/or contains almost 45 000 probe sets representing 439 000 generate tumor cells with cellular mechanisms enabling mRNA transcripts derived from B33 000 well-substan- escape from viral killing. We have shown this to be true at tiated human genes. This was utilized to profile the MOIs as high as several tens or hundreds. This potential mRNA, and thus gene-expression differences, between the of tumor resistance to oncolytic viral therapy has not, to parent cells before oncolytic HSV treatments and our knowledge, been previously reported in the literature. the generated resistant cells after serial treatments of In order to ascertain potential mechanisms involved in the virus. this viral resistance, gene-expression patterns comparing Analysis revealed a total of 74 commonly up- or down- parent cells with resistant cells were examined via regulated genes with changes 42.5-fold between the microarray analysis after total RNA was extracted from parental and resistant cell lines of two common cancers each cell group. cDNA microarray GeneChip technique is (Table 1). These genes comprised mostly enzymes, an increasingly popular technology for genome-wide transporters and transcription regulators. Their most expression profiling of cellular responses to many statistically significant roles were in cell morphology, environmental stimuli, including infections with a variety cellular development, cellular movement, cellular growth of viruses.19,22–25 For these experiments, we used cDNA and proliferation and cell-to-cell signaling and interac- GeneChip microarray Human Genome U133A, which tion. Cancer was the most statistically significant disease
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 44
Figure 3 Cell signaling pathway analysis involving common genes. Pathways with bars above the line have a P-value of X0.05. Analysis revealed six statistically significant pathways including PXR/RXR activation, IL-4 activation and acute-phase response signaling, as well as observed pathway involvements including xenobiotic metabolism signaling and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling.
to which commonly changed genes were linked, more receptors for HSV, and have proposed that binding of specifically functions related to tumorigenesis, transfor- HSV to cells might require interaction with heparan sulfate mation, growth, migration, detachment, attachment, as well as with some other cell surface molecule.26–28 It has binding, invasion and survival of cancer cells. The list also been suggested that interaction with heparin sulfate seems consistent and relevant to the phenomenon of viral and FN1, a member of the integrin class of cell surface oncolysis being studied. From a physiologic system receptors that has specific binding domains for both cell development viewpoint, functions involving cell-mediated surface heparan sulfate and for receptors of the integrin immune responses were the second most statistically superfamily, may affect HSV attachment and ability to significant. Specifically, immune responses affecting infect.27 Our microarray analysis showed decreased FN1 costimulation, quantity, selection of immune cells and expression in resistant cells compared with sensitive cells, aggregation appear to be involved. which may lead to less viral attachment. This may be one Network and pathway analysis revealed more thought- of the mechanisms that cancer cells utilize in order to resist provoking information. Six major networks involving the infection by herpes viruses. 74 genes commonly upregulated and downregulated Underexpression of FN1 is also involved in the hepatic between the two cell lines were identified, with 58 genes fibrosis/stellate cell activation pathway, which was shown deemed network eligible (Table 2 and Figure 4). The to be a statistically significant pathway in our analysis. network, including genes involved in cancer, cell mor- This decreased expression appears to be induced by phology and cellular compromise, achieved the highest increased interferon-g production, also known to cause score by IPA. Other networks involved organismal viral resistance. functions, hepatic system development and function and carbohydrate metabolism; cellular development and Another possible mechanism of resistance by these cells cellular growth and proliferation; cancer, cellular move- is hindering viral entry ment and cellular growth and proliferation; cancer, It is widely accepted that the main method of entry by energy production and small molecule biochemistry; and herpes virus is through direct membrane fusion and free radical scavenging. endocytosis.29 However, it has recently been proposed In addition to network analysis, IPA identified 52 genes that herpes viruses may also utilize other means. Clement eligible for pathways analysis. Moreover, six signaling et al.30 proposed a phagocytosis-like uptake in HSV entry. pathways were statistically significant: PXR/PXR activa- Our own analysis suggests that a caveolar-mediated tion; hepatic fibrosis/stellate cell activation; PPARa/ endocytotic pathway may also be exploited by herpes RXRa activation; IL-4 signaling; caveolar-mediated endo- viruses. Caveolar-mediated endocytosis plays a role in cytosis; and acute-phase response signaling (Figure 3). various cellular processes such as endocytosis, cellular signaling and lipid recycling.31 We detected two common Hindrance of viral attachment was one of the genes involved in this pathway: the genes INSR mechanisms by which generated cells resisted herpes and FLNB (filamin). INSR, which was underexpressed virus infection in our study, has been shown to be concentrated around Several papers have recently shown that heparan sulfate necks of caveolar membranes.32 On the other hand, moieties of cell surface proteoglycans are known to serve as FLNB, otherwise known as filamin, is also involved in
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 45
Figure 4 Network and pathway schema. (a) Network 1 includes 21 focus genes with top functions associated with cancer, cell morphology and cellular compromise. (b) Pathway schema of IL-4 signaling, in which SMAD3 is overexpressed and corticosteroid receptor (CR) is underexpressed, leading to overall increased cell proliferation and survival. Red-colored genes are overexpressed as compared with parental line; green are underexpressed.
caveolar-mediated endocytosis and was overexpressed in In addition, the PXR/RXR activation pathway plays a our study. Underexpression of INSR and overexpression fundamental role in regulating expression of a critical set of filamin suggest that, if indeed herpes viruses exploit this of protective gene products involved in xenobiotic and method of cell entry, the resistant cells have developed endobiotic metabolism.31 The MRP2 gene , which directly ways to prevent caveolar-mediated endocytosis of herpes regulates transport of xenobiotics (such as viruses) and viruses. The herpes virus may also have originally used other organic compounds in this pathway, was under- this pathway for entry into cells; or else, as the resistance expressed in our experiment. This suggests that another of generated cells increased, the virus exploited other mechanism by which cells have formed resistance to means of access. This implies that these cells are viruses is by decreasing their transport and metabolism, constantly evolving to try to prevent viral entry. further hindering viral entry.
Cancer Gene Therapy Gene expression in therapy-resistant cancer T-J Song et al 46 Viral replication may also be hindered by resistant cells apoptosis and mediates the immune response to the Herpes viruses are known to infect cells best during G1/S invading pathogen. In our study the underexpression of phase.33 This contributes to the preferential infection of the GFR gene eventually led to less NF-kB activation via rapidly dividing cells, such as cancer cells, as there is more a decrease in Ik-B expression. GFR-dependent activa- opportunity to replicate during this phase with every cell tion of NF-kB also involves phosphorylation of IkK division cycle. Our study indicated that genes involved in by the mitogen-activated protein kinase kinase kinase the G1/S-phase regulation are altered; in particular, a (MAPKKK) COT and its upstream enzyme AKT. decrease in cyclin-dependent kinase 4/6 by overexpression Of note, too, is the SOCS2 gene, also known as of SMAD3 (SMAD family member 3) appears to inhibit STAT-induced STAT inhibitor (SSI), which is a cytokine- progression into late G1 and S phase, possibly hindering inducible negative regulator of cytokine signaling. viral replication within these cells. Depletion of SOCS1 impaired G207 replication more than 10-fold,34 and SOCS1 mimetics protected against Alteration in pathways associated with increase in poxvirus infections.35,36 Thus, downregulation of SOCS2 tumor cell survival and proliferation via the IL-4 also seems to be a marker of potential resistance to pathway confers viral resistance oncolytic HSV therapy. Our analysis revealed an increase in IL-4R levels. JAK1 (Janus kinase 1) activation by IL-4Ra binding leads to ‘Universal genes’ may be associated with more increased cell proliferation, whereas the transcriptional aggressive and invasive tumors regulator STAT6 (signal transducer and activator of Many of the genes over- or under-expressed in our study transcription 6) is able to induce more IL-4Ra production may be ‘universal genes’ associated with more aggressive (Figure 4).31 Of note, STAT6 is inhibited by the and invasive tumors. Many upregulated genes are corticosteroid receptor, which our analysis revealed to associated with cancer development and progression, be underexpressed, further confirming a probable increase such as AIM-1,37 MICAL2,38 TMEPAI39 and Hif1a.40,41 in STAT6 activation by IL-4. Some of the downregulated genes in our study are also associated with tumorigenesis, such as DUSP9/MAPK4,42 43,44 45 46 Generated resistant cells appeared to have increased SEPP1, CSTA and SOCS2. This begs the ques- acute-phase responses to viral infection, compared with tion of whether cell lines with increased resistance to sensitive cells oncolytic viral therapy are also generally resistant to other Genes altered in our analysis and involved in these forms of conventional treatment, and thus potentially pathways include GCR or NRC1 (underexpressed), more aggressive. suppressor of cytokine signaling SOCS (overexpressed) In this study, we show the potential of certain tumors to and FN1 (underexpressed). The acute-phase response is exhibit resistance to oncolytic viral therapy, which has not a rapid inflammatory response that provides protection been previously reported in the literature. The concept of against microorganisms using nonspecific defense cancer cells acquiring viral resistance with multiple mechanisms.31 Positive acute-phase response proteins administration of virus is important, as it must be have general functions in opsonization and trapping factored into the development and progress of clinical of microorganisms, complement activation, neutralizing trials as well as clinical application of oncolytic viral enzymes and modulating the immune response. In this therapies. Further investigation of the mechanisms pathway, infection, inflammation, tissue injury and associated with the emergence of oncolytic viral resistance malignant disorders cause increased IL-6, IL-1 and is needed in order to develop strategies to overcome this tumor necrosis factor-a release. GCR increases transcrip- potential limitation. The current study provides a list of tion of the A2M gene in nuclei, a gene involved in IL-6 genes and pathways for further detailed analysis. This list signaling. IL-6 subsequently binds to JAK2, leading to may also be studied as patient selection criteria for trials increased STAT3 expression, thus increasing plasma of oncolytic viral therapy. Our study provides a frame- concentration of certain chemicals and complement work for the observation of possible cellular events, as molecules in the plasma. Overexpression of SOCS leads well as potential biologic and molecular targets, to to less inhibition of JAK2, thereby enhancing this overcome oncolytic viral resistance. response pathway. Another example of altered inflammatory responses in our study was the decreased activation of the PPARa/ Conflict of interest RXRa pathway leading to an overall decrease in IkBa and, subsequently, to anti-inflammatory effects. The The authors declare no conflict of interest. genes we detected that were involved in this pathway include INSR and PLC, both of which were under- Acknowledgements expressed. NF-kB transcription factors are key regulators of gene This work was supported by Grants R01CA75416 and expression, culminating in response to stress and the R01CA72632 from the National Institutes of Health (to development of innate and acquired immunity.31 Many YF), MBC-99366 from the American Cancer Society (to viruses, including HSV, activate NF-kB. The activation of YF) and a grant from the Flight Attendant Medical NF-kB promotes viral replication, prevents virus-induced Research Institute (FAMRI; to YF).
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