EGFR exon-level biomarkers of the response to bevacizumab/erlotinib in non-small cell lung cancer

Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.

Sarcoid-derived fibroblasts: links between genomic instability, energy metabolism and senescence.

Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated β-galactosidase (SA-β-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.

TYPE I INSULIN-LIKE GROWTH FACTOR RECEPTOR TYROSINE KINASE AS A MOLECULAR TARGET IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoid malignancy representing 5-10% of all non-Hodgkin’s lymphomas. It is distinguished by the t(11;14)(q13;q32) chromosomal translocation that juxtaposes the proto-oncogene CCND1, which encodes cyclin D1 at 11q13 to the IgH gene at 14q32. MCL patients represent about 6% of all new cases of Non-Hodgkin’s lymphomas per year or about 3,500 new cases per year. MCL occurs more frequently in older adults – the average age at diagnosis is the mid-60s with a male-to-female ratio of 2-3:1. It is typically characterized by the proliferation of neoplastic B-lymphocytes in the mantle zone of the lymph node follicle that have a prominent inclination to disseminate to other lymphoid tissues, bone marrow, peripheral blood and other organs. MCL patients have a poor prognosis because they develop resistance/relapse to current non-specific therapeutic regimens. It is of note that the exact molecular mechanisms underlying the pathogenesis of MCL are not completely known. It is reasonable to anticipate that better characterization of these mechanisms could lead to the development of specific and likely more effective therapeutics to treat this aggressive disease. The type I insulin-like growth factor receptor (IGF-IR) is thought to be a key player in several different solid malignancies such as those of the prostate, breast, lung, ovary, skin and soft tissue. In addition, recent studies in our lab showed evidence to support a pathogenic role of IGF-IR in some types of T-cell lymphomas and chronic myeloid leukemia. Constitutively active IGF-IR induces its oncogenic effects through the inhibition of apoptosis and induction of transformation, metastasis, and angiogenesis. Previous studies have shown that signaling through IGF-IR leads to the vi activation of multiple signaling transduction pathways mediated by the receptor-associated tyrosine kinase domain. These pathways include PI3K/Akt, MAP kinase, and Jak/Stat. In the present study, we tested the possible role of IGF-IR in MCL. Our results demonstrate that IGF-IR is over-expressed in mantle cell lymphoma cell lines compared with normal peripheral blood B- lymphocytes. Furthermore, inhibition of IGF-IR by the cyclolignan picropodophyllin (PPP) decreased cell viability and cell proliferation in addition to induction of apoptosis and G2/M cell cycle arrest. Screening of downstream oncogenes and apoptotic proteins that are involved in both IGF-IR and MCL signaling after treatment with PPP or IGF-IR siRNA showed significant alterations that are consistent with the cellular changes observed after PPP treatment. Therefore, our findings suggest that IGF-IR signaling contributes to the survival of MCL and thus may prove to be a legitimate therapeutic target in the future.

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

E2F1 AND TUMOR SUPPRESSION: THE ROLE OF p21, miRNAS, AND THE DNA DAMAGE RESPONSE

E2F1 is a multi-faceted protein that has roles in a number of important cellular processes including cell cycle regulation, apoptosis, proliferation, and the DNA damage response (DDR). Moreover, E2F1 has opposing roles in tumor development, acting as either a tumor suppressor or an oncogene depending on the context. In human cancer, E2F1 is often deregulated through aberrations in the Rb-p16INK4a-cyclin D1 pathway. In these studies we examined three mechanisms by which E2F1 might mediate its tumor suppressive properties: p21-induced senescence, miRNAs, and the DNA damage response. We found that E2F1 acts as a tumor suppressor in response to ras activation through a non-apoptotic mechanism requiring ARF and p53, but not p21. However, p21-loss inhibited two-stage chemical carcinogenesis in FVB mice. In response to E2F1 overexpression, we found that 22 miRNAs are differentially regulated in mouse epidermis, including let-7a, let-7c, and miR-301. Additionally, regulation of miR-301 involves binding of E2F1 to its promoter. Finally, our data indicate a role for E2F1 at sites of DNA damage requiring E2F1’s phosphorylation at serine 31 which may involve DNA repair. Further, this role in the DDR may affect tumor aggressiveness and multiplicity. In all, we have explored three mechanisms for E2F1-induced tumor suppression and identified E2F1’s role in the DNA damage response as a likely contributor to this phenomenon.

Analyzing the Role of αvβ8 Integrin in Glioma-Induced Angiogenesis

In this study we aimed to determine the functional roles for αvβ8 integrin in astrocytoma-induced angiogenesis. These studies originate from our analyses of αvβ8 integrin in developmental brain angiogenesis. αv and β8 knockout (KO) mice develop brain-specific vascular phenotypes that resemble vascular pathologies observed in the malignant astrocytoma, glioblastoma multiforme (GBM). Indeed, a murine xenograft model of astrocytoma suggested a role for the integrin in glioma-induced angiogenesis. Primary mouse astroglia were cultured from wild type (WT) and β8 KO neonates and were immortalized (HPV:E6/E7) and transformed (HRas:G12V). WT and β8 KO transformed astroglia were intracranially injected into athymic mice. WT tumors displayed pathological features of grade III astrocytomas, whereas β8 KO tumors resembled grade IV GBMs. KO tumors contained widespread edema and hemorrhage as well as pathological angiogenesis, as assessed by quantitation of microvascular density and blood vessel morphology. Additionally, exogenous expression of β8 integrin in β8 KO transformed astroglia resolved the pathologies observed in KO tumors giving further credence to the idea that loss of αvβ8 integrin expression correlates with tumorigenic potential of oncogene-transformed astroglia. To compliment our mouse model, several established human glioma cell lines were characterized for expression of αvβ8 integrin protein. Some of the cell lines displayed low expression of αvβ8 integrin, whereas others showed high levels, as compared to non-malignant human astrocytes. Intracranial implantation of high and low β8 integrin-expressing human glioma cell lines resulted in tumors exhibiting similar phenotypes to those observed in the mouse model; low expressers were marked by vascular pathologies indicative of β8 KO mouse tumors. Upon overexpression of β8 integrin in a low β8 integrin-expressing human glioma cell line, angiogenic pathologies were largely resolved. Moreover, intracranially injected αvHI- and αvLO-sorted GBM stem cells (GSCs) resulted in significantly different tumor sizes, where those GSCs endogenously expressing low levels of αv integrin formed two to three fold larger tumors. Furthermore, lentiviral knockdown of β8 integrin in transformed human astrocytes formed tumors that strikingly recapitulated the characteristics of the murine β8-/- tumors, exhibiting a significant increase in microvascular density leading to decreased overall survival. A paracrine mechanism was discovered involving endothelial cell homeostatic control governed by canonical TGFβ signaling initiated by αvβ8 integrin’s role in the latent cytokine’s activation. Diminished TGFβ signaling in tumor-associated endothelial cells promoted increased angiogenesis and decreased overall survival as a result of αvβ8 integrin’s loss on the tumor cell. Collectively, these data suggest an important functional role for αvβ8 integrin in glioma angiogenesis.

MCAM/MUC18 REGULATES MELANOMA PROGRESSION BY MODULATING THE EXPRESSION OF ID-1

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression utilizing lentivirus-incorporated small hairpin RNA, in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, Inhibitor of DNA Binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3. Interestingly, ATF-3 was upregulated by 6.9 fold in our cDNA microarray analysis following MUC18 silencing. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing, while mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18-silenced cells. These Data suggest that MUC18 silencing promotes inhibition of Id-1 expression by increasing ATF-3 expression and binding to the Id-1 promoter. Rescue of MUC18 reverted the expression of Id-1 and ATF-3, thus validating that they are not off-target effects of MUC18. To further assess the role of Id-1 in melanoma invasion and metastasis, we overexpressed Id-1 in MUC18-silenced cells. Overexpression of Id-1 in MUC18-silenced cells resulted in increased cell invasion, as well as increased expression and activity of MMP-2. Our data further reveal that Id-1 regulates MMP-2 at the transcriptional level through Sp1 and Ets-1. This is the first report to demonstrate that MUC18 does not act exclusively in cell adherence, but is also involved in cell signaling that regulates the expression of genes, such as Id-1 and ATF-3, thus contributing to the metastatic melanoma phenotype.

NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Intra-hippocampal administration of the VEGF receptor blocker PTK787/ZK222584 impairs long-term memory.

A number of studies have established a role for vascular endothelial growth factor (VEGF) in angiogenesis. Recent reports have shown that VEGF overexpression in the hippocampus improves learning and memory and is associated with enhanced neurogenesis. PTK787/ZK222584 (PTK/ZK) is a reported inhibitor of VEGFR signaling that is currently being tested for its effects on lung and colon cancer. However, the influence of this drug on cognition has not been examined. In the present study, we questioned if post-training administration of PTK/ZK influences hippocampus-dependent memory. When administered to rats immediately following massed training in the Morris water maze, PTK/ZK impaired spatial memory retention tested 48 h later. This impairment was evidenced by increased latency to the hidden platform and fewer platform crossings. However, this impairment was not associated with a change in neurogenesis during this time frame. PTK/ZK infusion did not reduce VEGFR or AKT phosphorylation, but increased the phosphorylation of ERK. These studies suggest that VEGFR inhibitors such as PTK/ZK may negatively influence cognition.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x>10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.