Functional oncogene signatures guide rationally designed combination therapies to synergistically induce breast cancer cell death.

A critical first step in the personalized approach to cancer treatment is the identification of activated oncogenes that drive each tumor. The Identification of driver oncogenes on a patient-by-patient basis is complicated by the complexity of the cancer genome and the fact that a particular genetic alteration may serve as a driver event only in a subset of tumors that harbor it. In this study, we set out to identify the complete set of functional oncogenes in a small panel of breast cancer cell lines. The cell lines in this panel were chosen because they each contain a known receptor tyrosine kinase (RTK) oncogene. To identify additional drivers, we integrated functional genetic screens with copy number and mutation analysis, and cancer genome knowledge databases. The resulting functional oncogene signatures were able to predict responsiveness of cell lines to targeted inhibitors. However, as single agents, these drugs had little effect on clonogenic potential. By contrast, treatment with drug combinations that targeted multiple oncogenes in the signatures, even at very low doses, resulted in the induction of apoptosis and striking synergistic effects on clonogenicity. In particular, targeting a driver oncogene that mediates AKT phosphorylation in combination with targeting the anti-apoptotic BCL2L1 protein had profound effects on cell viability. Importantly, because the synergistic induction of cell death was achieved using low levels of each individual drug, it suggests that a therapeutic strategy based on this approach could avoid the toxicities that have been associated with the combined use of multiple-targeted agents.

Two members of the TRiC chaperonin complex, CCT2 and TCP1 are essential for survival of breast cancer cells and are linked to driving oncogenes.

Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer.

RAS/MEK-independent gene expression reveals BMP2-related malignant phenotypes in the Nf1-deficient MPNST.

Malignant peripheral nerve sheath tumor (MPNST) is a type of soft tissue sarcoma that occurs in carriers of germline mutations in Nf1 gene as well as sporadically. Neurofibromin, encoded by the Nf1 gene, functions as a GTPase-activating protein (GAP) whose mutation leads to activation of wt-RAS and mitogen-activated protein kinase (MAPK) signaling in neurofibromatosis type I (NF1) patients' tumors. However, therapeutic targeting of RAS and MAPK have had limited success in this disease. In this study, we modulated NRAS, mitogen-activated protein/extracellular signal-regulated kinase (MEK)1/2, and neurofibromin levels in MPNST cells and determined gene expression changes to evaluate the regulation of signaling pathways in MPNST cells. Gene expression changes due to neurofibromin modulation but independent of NRAS and MEK1/2 regulation in MPNST cells indicated bone morphogenetic protein 2 (Bmp2) signaling as a key pathway. The BMP2-SMAD1/5/8 pathway was activated in NF1-associated MPNST cells and inhibition of BMP2 signaling by LDN-193189 or short hairpin RNA (shRNA) to BMP2 decreased the motility and invasion of NF1-associated MPNST cells. The pathway-specific gene changes provide a greater understanding of the complex role of neurofibromin in MPNST pathology and novel targets for drug discovery.

Racial differences in oncogene mutations detected in early-stage low-grade endometrial cancers.

OBJECTIVE: To describe the pattern and frequency of oncogene mutations in white and African American women with endometrial cancer and to determine if racial differences in oncogene mutations exist among women with pathologically similar tumors. METHODS: Patients with endometrial cancer from a large urban hospital were identified through medical records, and representative formalin-fixed paraffin-embedded tumor blocks were retrieved. The study sample included 150 patients (84 African Americans) who underwent total abdominal hysterectomy for endometrial cancer. The Sequenom MassARRAY system and the OncoCarta Assay version 1.0 (Sequenom) were used to test for 238 mutations in 19 common oncogenes. The χ(2) test and the Fisher exact test were used to assess differences in distribution of variables by race and oncogene mutation status. RESULTS: There were 20 mutations identified in 2 oncogenes (PIK3CA and KRAS) in tumors from 19 women (12.7%). Most of the mutations were found in PIK3CA (16/20). Thirteen percent of endometrioid tumors harbored mutations (11 PIK3CA and 2 KRAS) as did 29% of the malignant mixed Mullerian tumors (3 PIK3CA and 1 KRAS). There were no observed mutations in serous, clear cell, or mucinous tumor types. Among low-grade endometrioid cancers, tumors from African American patients were significantly associated with harboring either a KRAS or PIK3CA mutation (P = 0.04), with 7 PIK3CA mutations and all 4 KRAS mutations identified in African American women. CONCLUSIONS: This study provides preliminary evidence that oncogene mutation frequency of some subtypes of histologically similar endometrial carcinoma differ by race. Additional studies are needed to further explore this phenomenon in patients with endometrial carcinoma.

Oncogene Mutation Survey in MPNST Cell Lines Enhances the Dominant Role of Hyperactive Ras in NF1 Associated Pro-Survival and Malignancy.

Malignant peripheral nerve sheath tumors (MPNST) are a type of soft tissue sarcoma that can be associated with germline mutations in Neurofibromatosis type 1 (NF1) or may occur sporadically. Although the etiology of MPNST is poorly understood, it is clear that a loss of function of the NF1 gene, encoding a Ras-GAP, is an important factor in the tumorigenesis of the inherited form of MPNST. Tumor latency in NF1 patients suggests that additional mutational events are probably required for malignancy. In order to define oncogene mutations associated with 5 MPNST cell lines, we assayed the 238 most frequent mutations in 19 commonly activated oncogenes using mass spectroscopy-based analysis. All 238 mutation sites in the assayed oncogenes were determined to harbor only wild-type sequences. These data suggest that hyperactive Ras resulting from the loss function of neurofibromin may be sufficient to set up the direction of malignant transformation of Schwann cells to MPNST.

Frequency and type of epidermal growth factor receptor mutations in African Americans with non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict response to tyrosine kinase inhibitors. Mutations occur more commonly in never smokers and East Asians, but there are conflicting reports on the frequency of EGFR mutations in tumors from African Americans. METHODS: Tumors from 67 African American and 77 white participants in previous case-control studies of lung cancer were selected to determine EGFR mutational status. Mutation analysis was performed using the Sequenom mass array analyzer (Sequenom, San Diego, CA). RESULTS: Overall, 13.9% of the study population carried an EGFR mutation. EGFR mutations occurred in 11.9% of tumors from African Americans compared with 15.6% in whites (p = 0.53). All mutations found in African Americans were deletions in exon 19. The majority of mutations were found in nonsmokers among both African Americans (7/8) and whites (8/12). CONCLUSION: These results indicate that African Americans with NSCLC harbor somatic EGFR mutations at a frequency similar to whites with NSCLC. Thus, clinicians should not use race as a clinical decision parameter for the use of EGFR-tyrosine kinase inhibitors.

HER-2 signaling, acquisition of growth factor independence, and regulation of biological networks associated with cell transformation.

Activated oncogenes are the dominant drivers of malignant progression in human cancer, yet little is known about how the transformation from proto-oncogene to activated oncogene drives the expression of transformed phenotypes. An isogenic model of HER-2-mediated transformation of human mammary epithelial cells was used along with HER-2-amplified human breast cancers to investigate how HER-2 activation alters its properties as a signaling molecule and changes the networks of HER-2-regulated genes. Our results show that full oncogenic activation of HER-2 is the result of a transition in which activated HER-2 acquires dominant signaling properties that qualitatively alter the network of genes regulated by the activated oncogene compared with the proto-oncogene. Consequently, gene expression programs related to invasion, cell stress, and stemness become regulated by HER-2 in a manner not observed in nontransformed cells, even when HER-2 is overexpressed. Our results offer novel insights into biological processes that come under the control of HER-2 after it acquires full oncogenic potential.

EGFR/Met association regulates EGFR TKI resistance in breast cancer.

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.

Methylation-associated silencing of SFRP1 with an 8p11-12 amplification inhibits canonical and non-canonical WNT pathways in breast cancers.

Recently, we analysed the 8p11-12 genomic region for copy number and gene expression changes in a panel of human breast cancer cell lines and primary specimens. We found that SFRP1 (Secreted frizzled related protein 1) is frequently under expressed even in breast tumours with copy number increases in this genomic region. SFRP1 encodes a WNT signalling antagonist, and plays a role in the development of multiple solid tumour types. In this study, we analysed methylation-associated silencing of the SFRP1 gene in breast cancer cells with the 8p11-12 amplicon, and investigated the tumour suppressor properties of SFRP1 in breast cancer cells. SFRP1 expression was markedly reduced in both the breast cancer cell lines and primary tumour specimens relative to normal primary human mammary epithelial cells even when SFRP1 is amplified. Suppression of SFRP1 expression in breast cancer cells with an SFRP1 gene amplification is associated with SFRP1 promoter methylation. Furthermore, restoration of SFRP1 expression suppressed the growth of breast cancer cells in monolayer, and inhibited anchorage independent growth. We also examined the relationship between the silencing of SFRP1 gene and WNT signalling in breast cancer. Ectopic SFRP1 expression in breast cancer cells suppressed both canonical and non-canonical WNT signalling pathways, and SFRP1 expression was negatively associated with the expression of a subset of WNT responsive genes including RET and MSX2. Thus, down-regulation of SFRP1 can be triggered by epigenetic and/or genetic events and may contribute to the tumourigenesis of human breast cancer through both canonical and non-canonical WNT signalling pathways.

Angiogenesis gene expression in mouse uterus during the common pathway of parturition.

OBJECTIVE: The objective of the study was to investigate changes in the expression of angiogenesis-related genes during the common terminal pathway of parturition including spontaneous labor at term, as well as preterm labor (PTL), induced by either bacteria or ovariectomy. STUDY DESIGN: Preterm pregnant mice (14.5 days of gestation) were treated with the following: (1) intrauterine injection of media; (2) intrauterine injection of heat-inactivated Escherichia coli; (3) ovariectomy; and (4) sham operation. Tissues from mice at term (19.5 days of gestation) were collected at term not in labor, term in labor, and 12 hours postpartum. Angiogenesis-related gene expression levels were quantitated by the measurement of specific mRNAs in uterine tissue by RT-qPCR and analyzed by repeated-measures analysis of variance. RESULTS: The following results were found: (1) microarray analysis of the uterine transcriptome indicated an enrichment for the gene ontology category of angiogenesis in bacteria-induced PTL samples (P < or = .093); (2) several genes related to angiogenesis demonstrated significantly increased expression in samples in either term spontaneous labor or preterm labor; and (3) qRT-PCR measurements demonstrated that spontaneous term labor and preterm labor induced by either bacteria or ovariectomy all substantially increased the expression of multiple angiogenesis-related genes (P < or = .0003; Angpt2, Ctgf, Cyr61, Dscr1, Pgf, Serpine1, Thbs1, and Wisp 1). CONCLUSION: Spontaneous labor at term, as well as pathologically induced preterm labor, all result in greatly increased expression of angiogenesis-related genes in the uterus.

Transforming properties of TC-1 in human breast cancer: interaction with FGFR2 and beta-catenin signaling pathways.

Breast cancer development is associated with gene amplification and over expression that is believed to have a causative role in oncogenesis. Previous studies have demonstrated that over expression of TC-1(C8orf4) mRNA occurs in approximately 50% of breast cancer cell lines and primary tumor specimens. Here, we show that TC-1 has transforming properties in human mammary epithelial (HME) cells and its expression is mechanistically linked to FGFR signaling cascades. In vitro experiments demonstrate that TC-1 over expression mediates both anchorage-independent and growth factor-independent proliferation of HME cells. TC-1 was down regulated by the FGFR inhibitor PD173074 in the breast cancer cell line SUM-52 that also has an FGFR2 gene amplification and over expression. Furthermore, forced expression of FGFR2 in HME cells increased the level of expression of endogenous TC-1 mRNA. TC-1 has been implicated as a modulator of Wnt/beta-catenin signaling in 293 cells and in gastric cancer cells. However, while we did find increased expression of a subset of beta-catenin target genes in TC-1 over expressing cells, we did not find an association of TC-1 with global expression of beta-catenin target genes in our cells. Taken together, our data suggest that TC-1 over expression is transforming and may link with the FGFR pathway in a subset of breast cancer.

The transcriptome of the uterine cervix before and after spontaneous term parturition.

OBJECTIVE: This study was designed to identify genes differentially expressed in the human uterine cervix after spontaneous term labor. STUDY DESIGN: The transcriptome of cervical tissue was characterized using Affymetrix HG-U133 plus 2 microarrays. Samples were collected from patients at term not in labor (n = 7) and after spontaneous labor (n = 9). Microarray statistical analysis included robust multiarray average, reduction of invariant probes, and permutation analysis for differential expression. Real-time quantitative reverse transcriptase-polymerase chain reaction assays of selected genes were performed on a new set of samples from term patients without labor (n = 10) and patients after spontaneous labor (n = 9). RESULTS: (1) The cervical transcriptome of term patients without labor was dramatically different from that of patients who underwent labor; (2) unique genes (n = 1192) were differentially expressed in the cervical tissue from patients after spontaneous labor, compared with that of the term patients without labor (false discovery rate less than 0.05, absolute fold change greater than 2); (3) Gene Ontology analysis indicated that multiple "Biological Process" categories were enriched, including "response to biotic stimulus," "apoptosis," "epidermis development," and "steroid metabolism"; (4) of major interest, genes involved in neutrophil chemotaxis were dramatically up-regulated in specimens from women after spontaneous labor; (5) real-time quantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of interleukin-8, interleukin-6, and vascular endothelial growth factor in patients after spontaneous labor; and (6) Toll-like receptor-3 and Toll-like receptor-5 showed decreased gene expression in patients after spontaneous labor. This was confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. CONCLUSION: (1) Cervical dilatation in term labor is associated with a stereotypic gene expression pattern determined by microarray, which is characterized by overexpression of genes involved in neutrophil chemotaxis, apoptosis, extracellular matrix regulation, and steroid metabolism; (2) Toll-like receptor-3 and Toll-like receptor-5 are differentially regulated during spontaneous parturition at term; and (3) this study provides an unbiased and comprehensive description of the changes in the cervical transcriptome before and after spontaneous term labor.

Uterine transcriptomes of bacteria-induced and ovariectomy-induced preterm labor in mice are characterized by differential expression of arachidonate metabolism genes.

OBJECTIVE: The purpose of this study was to identify changes in gene expression that are associated with preterm labor induced by either bacteria or ovariectomy. STUDY DESIGN: Pregnant mice (14.5 days of gestation) were allocated to: (1) intrauterine injection of heat-inactivated Escherichia coli; (2) media alone; (3) ovariectomy; or (4) sham operation. The uterine transcriptome was studied with photolithographic, very short oligonucleotide-based microarrays, and arachidonate metabolism genes were assayed with quantitative reverse transcriptase-polymerase chain reaction. Significance was determined by analysis of variance. RESULTS: Microarray-based gene expression changes in the arachidonate metabolism pathway are associated globally with bacteria-induced preterm labor (P < or = .0031) and ovariectomy-induced preterm labor (P < or = .00036). Quantitative real-time reverse transcriptase-polymerase chain reaction measurements demonstrated that bacteria-induced preterm labor substantially increased the expression of genes involved in prostaglandin synthesis. In contrast, ovariectomy-induced preterm labor increased the expression of genes involved in lipoxin, leukotriene, and hydroxyeicosatetraenoic acid synthesis. CONCLUSION: Bacteria-induced and ovariectomy-induced preterm labor each express a different balance of genes that are required for the synthesis of prostaglandins, lipoxins, leukotrienes, and hydroxyeicosatetraenoic acids.

Human spontaneous labor without histologic chorioamnionitis is characterized by an acute inflammation gene expression signature.

OBJECTIVE: The purpose of this study was to identify which biological processes may be involved in normal labor. STUDY DESIGN: Transcriptional profiles for chorioamniotic membranes (n = 24) and blood (n = 20) were generated from patients at term with no labor (TNL) and in labor (TIL). RESULTS: Expression of 197 transcripts (P < or = .02) differentiated TIL and TNL chorioamniotic membrane samples. Gene Ontology analysis indicated that TIL samples had increased expression of multiple chemokines and transcripts associated with neutrophil and monocyte recruitment. Microarray results were verified using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) with independent samples. Transcriptional profiles from blood RNA revealed no Gene Ontology category enrichment of discriminant probe sets. CONCLUSION: Labor induces gene expression changes consistent with localized inflammation, despite the absence of histologically detectable inflammation.

Expression of bone morphogenetic protein 2 in normal spontaneous labor at term, preterm labor, and preterm premature rupture of membranes.

OBJECTIVE: Genome-wide screening studies of the chorioamniotic membranes unexpectedly identified an increase in the expression of bone morphogenetic protein 2 in spontaneous labor at term. The objective of this study was to determine whether bone morphogenetic protein 2 messenger RNA and protein expression are altered in the chorioamniotic membranes of patients with term labor, preterm labor, and preterm premature rupture of membranes. STUDY DESIGN: Chorioamniotic membranes were obtained from patients at term (with and without labor), with preterm labor (with and without histologic chorioamnionitis), and with preterm premature rupture of membranes (with and without histologic chorioamnionitis). The expression of bone morphogenetic protein 2 was studied by real-time quantitative reverse transcriptase-polymerase chain reaction (n = 88) and immunohistochemistry (n = 124). Nonparametric statistics were used for analysis. Primary amnion cells obtained from women at term not in labor were treated with bone morphogenetic protein 2 to examine whether there was increased prostaglandin E2 expression. RESULTS: The median bone morphogenetic protein 2 messenger RNA and protein expression were significantly higher in the membranes of patients with spontaneous labor at term than in those of patients not in labor at term (P < .001 for both). Bone morphogenetic protein 2 messenger RNA and protein expression were increased in patients with preterm labor with histologic chorioamnionitis than in those without histologic chorioamnionitis (P < .05 and P < .001, respectively). There was no difference in bone morphogenetic protein 2 messenger RNA and protein expression in patients with preterm premature rupture of membranes, regardless of chorioamnionitis (P = .13 and P = .08, respectively). There was a correlation between bone morphogenetic protein 2 and cyclooxygenase 2 protein expression in chorioamniotic membranes (R = .34; P < .001). CONCLUSION: Bone morphogenetic protein 2 messenger RNA and protein expression are increased in the chorioamniotic membranes of patients with spontaneous labor at term and patients with preterm labor associated with histologic chorioamnionitis. Its expression pattern and biologic effects strongly suggest that bone morphogenetic protein 2 is involved in human parturition.

Molecular profiling of human hepatocellular carcinoma defines mutually exclusive interferon regulation and insulin-like growth factor II overexpression.

Molecular subtyping of human hepatocellular carcinoma (HCC) with potential mechanistic and therapeutic impact has not been achieved thus far. We have analyzed the mRNA expression patterns of 43 different human HCC samples and 3 HCC cell lines in comparison with normal adult liver using high-density cDNA microarrays. Two main groups of HCC, designated group A (65%) and group B (35%), were distinguished based on clustering of the most highly varying genes. Group A HCCs were characterized by induction of a number of interferon (IFN)-regulated genes, whereas group B was characterized mainly by down-regulation of several apoptosis-relevant and IFN-regulated genes. The number of apoptotic tumor cells and tumor-infiltrating lymphocytes was significantly higher in tumors of group A as compared with those of group B. Based on the expression pattern, group B was further subdivided into two subgroups, designated subgroup B1 (6 of 43 tumors, 14%) and subgroup B2 (9 of 43 tumors, 21%). A prominent characteristic of subgroup B1 was high overexpression of insulin-like growth factor (IGF)-II. All tested HCC cell lines expressed equally high concentrations of IGF-II transcripts and co-segregated with group B1 in clustering. IGF-II overexpression and induction of IFN-related genes were mutually exclusive, even when analysis was extended to other cancer expression profile studies. Moreover, IFN-gamma treatment substantially reduced IGF-II expression in HCC cells. In conclusion, cDNA microarray analyses provided subtyping of HCCs that is related to intratumor inflammation and tumor cell apoptosis. This profiling may be of mechanistic and therapeutic impact because IGF-II overexpression has been linked to reduced apoptosis and increased proliferation and may be accessible to therapeutic intervention.

Oltipraz, 3H-1,2-dithiole-3-thione, and sulforaphane induce overlapping and protective antioxidant responses in murine microglial cells.

Oltipraz (OPZ) is a known inducer of glutathione S-transferases and a mechanism-based inhibitor of cytochrome P450 1A2. Given the detoxification characteristics of this compound, the transcriptional effects of OPZ, along with the related naturally occurring compounds 3H-1,2-dithiole-3-thione (D3T) and sulforaphane (SF), were examined by gene expression profiling in murine BV-2 microglial cells, a neuronal macrophage cell type that mediates inflammatory responses in the brain. We show that the three compounds generate largely overlapping transcriptional changes in genes that are associated with detoxification and antioxidant responses. In addition, induction of an antioxidant/detoxification response in the microglial cells by OPZ, D3T, or SF was also able to protect cells from H2O2 -induced toxicity and to attenuate the production of reactive oxygen species in response to lipopolysaccharide treatment of cells. These results show that OPZ, D3T, and SF activate overlapping changes in gene expression and that they can regulate detoxification/antioxidant responses in multiple cells types, including cell types known to have a role in the production of oxidative stress.