Bryony H. Lloyd

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

BackgroundNrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.ResultsKeap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001).ConclusionsExpression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.

Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumour cells.

The rodent S100-related calcium-binding protein, S100A4 induces metastasis in non-metastatic rat and mouse benign mammary cells and co-operates with benign-tumour-inducing changes in two transgenic mouse models, to yield metastatic mammary tumours. Co-transfection of the human gene for S100A4 with pSV2neo into the benign rat mammary cell line, Rama 37, yielded cells which expressed a low level of the endogenous S100A4 mRNA, and either high or undetectable levels of human S100A4 mRNA. The cells which expressed a high level of human S100A4 mRNA induced metastasis in the benign rat mammary cell line Rama 37 in an in vivo assay, whereas the cells which expressed an undetectable level of human S100A4 did not induce any detectable metastases. The primary tumours arising from the S100A4-expressing cells contained high levels of immunocytochemically-detected S100A4 and this high level of S100A4 and the metastatic potential were maintained when cells from a metastasis were re-injected into syngeneic rats. The results show that the human S100A4 possesses metastasis-inducing capabilities.

Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53.

Background:Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells.Methods:We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3.Results:LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity.Conclusion:This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients.

Single nucleotide polymorphism array analysis of uveal melanomas reveals that amplification of CNKSR3 is correlated with improved patient survival.

Metastatic death from uveal melanoma occurs almost exclusively with tumors showing monosomy of chromosome 3. However, approximately 5% of patients with a disomy 3 uveal melanoma develop metastases, and a further 5% of monosomy 3 uveal melanoma patients exhibit disease-free survival for >5 years. In the present study, whole-genome microarrays were used to interrogate four clinically well-defined subgroups of uveal melanoma: i) disomy 3 uveal melanoma with long-term survival; ii) metastasizing monosomy 3 uveal melanoma; iii) metastasizing disomy 3 uveal melanoma; and iv) monosomy 3 uveal melanoma with long-term survival. Cox regression and Kaplan-Meier survival analysis identified that amplification of the CNKSR3 gene (log-rank, P = 0.022) with an associated increase in its protein expression (log-rank, P = 0.011) correlated with longer patient survival. Although little is known about CNKSR3, the correlation of protein expression with increased survival suggests a biological function in uveal melanoma, possibly working to limit metastatic progression of monosomy 3 uveal melanoma cells.

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy.

Whats known on the subject? and What does the study add?

Localisation by in situ hybridisation of S100A4 (p9Ka) mRNA in primary human breast tumour specimens

Rodent S100A4 (p9Ka) induces a metastatic phenotype in benign rat mammary tumour cells and cooperates with the neu oncogene to produce metastatic tumours in a transgenic mouse model system. Human S100A4 possesses similar metastasis‐inducing properties. S100A4 mRNA is now sought in human breast tumour‐derived cell lines and tumour specimens. S100A4 mRNA is present in some cell lines derived from malignant breast cancers, but is not detectable in cells derived from benign breast tumours. In human tumour specimens, using in situ hybridisation, the mRNA for S100A4 is localised to the epithelial cells of carcinoma specimens, and in some normal breast specimens, to a stromal region surrounding the epithelial ducts. In carcinoma specimens, S100A4 mRNA is also found in the stromal region surrounding islands of cancer cells. For both the epithelial and stromal components, S100A4 mRNA is present at a higher level in carcinomas relative to benign breast tumour specimens. In general, there is a concordance between the S100A4 mRNA signal from the epithelial and stromal elements of the same carcinoma specimens. Using Northern blotting techniques, these results have been extended to a panel of 137 benign and malignant breast tumour specimens. The results show that S100A4 mRNA occurs in the more‐malignant, rather than in the more‐benign tumour specimens. Int. J. Cancer 86:219–228, 2000.

SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma.

Background:Extracapsular spread (ECS) in cervical lymph nodes is the single-most prognostic clinical variable in oral squamous cell carcinoma (OSCC), but diagnosis is possible only after histopathological examination. A promising biomarker in the primary tumour, alpha smooth muscle actin (SMA) has been shown to be highly prognostic, however, validated biomarkers to predict ECS prior to primary treatment are not yet available.Methods:In 102 OSCC cases, conventional imaging was compared with pTNM staging. SERPINE1, identified from expression microarray of primary tumours as a potential biomarker for ECS, was validated through mRNA expression, and by immunohistochemistry (IHC) on a tissue microarray from the same cohort. Similarly, expression of SMA was also compared with its association with ECS and survival. Expression was analysed separately in the tumour centre and advancing front; and prognostic capability determined using Kaplan–Meier survival analysis.Results:Immunohistochemistry indicated that both SERPINE1 and SMA expression at the tumour-advancing front were significantly associated with ECS (P<0.001). ECS was associated with expression of either or both proteins in all cases. SMA+/SERPINE1+ expression in combination was highly significantly associated with poor survival (P<0.001). MRI showed poor sensitivity for detection of nodal metastasis (56%) and ECS (7%). Both separately, and in combination, SERPINE1 and SMA were superior to MRI for the detection of ECS (sensitivity: SERPINE1: 95%; SMA: 82%; combination: 81%).Conclusion:A combination of SMA and SERPINE1 IHC offer potential as prognostic biomarkers in OSCC. Our findings suggest that biomarkers at the invasive front are likely to be necessary in prediction of ECS or in therapeutic stratification.

CYP2B6*6 is an independent determinant of inferior response to fludarabine plus cyclophosphamide in chronic lymphocytic leukemia

Fludarabine plus cyclophosphamide (FC) is the chemotherapy backbone of modern chronic lymphocytic leukemia (CLL) treatment. CYP2B6 is a polymorphic cytochrome P450 isoform that converts cyclophosphamide to its active form. This study investigated the possible impact of genetic variation in CYP2B6 on response to FC chemotherapy in CLL. Available DNA samples from the LRF CLL4 trial, which compared chlorambucil, fludarabine, and FC, were screened by TaqMan real-time polymerase chain reaction assays for CYP2B6 SNPs c.516G>T and c.785A>G, which define the most common variant allele (*6). Among the 455 samples successfully genotyped, 265 (58.2%), 134 (29.5%), and 29 (6.4%) were classified as *1/*1, *1/*6, and *6/*6, respectively. Patients expressing at least one *6 allele were significantly less likely to achieve a complete response (CR) after FC (odds ratio 0.27; P = .004) but not chlorambucil or fludarabine. Analysis of individual response indicators confirmed that this inferior response resulted from impaired cytoreduction rather than delayed hemopoietic recovery. Multivariate analysis controlling for age, gender, stage, IGHV mutational status, 11q deletion, and TP53 deletion/mutation identified CYP2B6*6 and TP53 mutation/deletion as the only independent determinants of CR attainment after FC. Our study provides the first demonstration that host pharmacogenetics can influence therapeutic response in CLL. This trial is registered as an International Standard Randomised Control Trial, number NCT 58585610 at www.clinicaltrials.gov.

Gene expression profiling in bladder cancer identifies potential therapeutic targets

Despite advances in management, bladder cancer remains a major cause of cancer related complications. Characterisation of gene expression patterns in bladder cancer allows the identification of pathways involved in its pathogenesis, and may stimulate the development of novel therapies targeting these pathways. Between 2004 and 2005, cystoscopic bladder biopsies were obtained from 19 patients and 11 controls. These were subjected to whole transcript-based microarray analysis. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Hypergeometric analysis was used to identify canonical pathways and curated networks having statistically significant enrichment of differentially expressed genes. Osteopontin (OPN) expression was validated by immunohistochemistry. Hierarchical clustering defined signatures, which differentiated between cancer and healthy tissue, muscle-invasive or non-muscle invasive cancer and healthy tissue, grade 1 and grade 3. Pathways associated with cell cycle and proliferation were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was markedly overexpressed in invasive cancer compared to healthy tissue. The present study contributes to a growing body of work on gene expression signatures in bladder cancer. The data support an important role for osteopontin in bladder cancer, and identify several pathways worthy of further investigation.

Abstract 5246: Genomic determinants of aggressive phenotype in oral SCC

Background: Oral squamous cell carcinoma (OSCC), the common form of HNSCC, affects over 600,000 cases worldwide per annum. We reviewed 561 consecutive cases in Liverpool 1 finding that the most predictive marker for death was extra-capsular spread (ECS) from cervical lymph nodes (p 2 . The presence of ECS predicted for recurrence, usually at the primary site, even when surgical margins were generous indicating that this aggressive phenotype is likely to be expressed in the primary tumour as much as in the metastatic cells. Hypothesis: We hypothesised that molecular pathways represented by global gene expression signatures would be detected in the samples associated with ECS. Methods: High density exon array expression analysis was performed on fresh frozen samples comprising T2 and T4 stage OSCC cases having no nodal involvement; positive nodes without ECS or positive nodes plus ECS picked longitudinally from a single centre series (n>200) with bias towards increasing the numbers with ECS. Contingency tests were performed to assess the significance of ECS associated pathways. Results and Discussion: Power analysis showed that 90% of all ≥ twofold differences in gene expression between node negative & ECS cases were significant. Affected pathways included: keratinocyte differentiation, ECM remodelling, chemokines & adhesion, TGF & WNT: cytoskeletal remodelling, regulation of actin cytoskeleton by rho GTPases & cell adhesion/ histamine H1 receptor signalling/loss of cell barrier (p values:7.7×10 −15 – 1.9×10 −6 ). A 29 gene signature able to discriminate the ECS from the non-ECS cases with 78% sensitivity and 86% specificity was found. Blocked differentiation of keratinocytes at an early (“stem cell”/ EMT) stage was suggested by the genes which included keratin 15, keratinocyte differentiation-associated protein, retinol binding protein 1, cellular serine peptidase inhibitor (Kazal type 6), and stratifin implicated in mesenchymal stem cells, basal, suprabasal expression and keratinocyte activation and differentiation. The signature may be connected with these tumours having poor prognosis. Conclusion: This is a significant and novel finding suggesting that the pattern of metastasis in regional lymph nodes can be predicted by molecular pathways prevalent in a small oral biopsy. 1. Rogers et al. Oral Oncol 2009;45(3):201-211 2. Shaw et.al. Head Neck 2010;32(6):714-722 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5246. doi:10.1158/1538-7445.AM2011-5246