ERCC1 isoform expression and DNA repair in non-small-cell lung cancer.

BACKGROUND: The excision repair cross-complementation group 1 (ERCC1) protein is a potential prognostic biomarker of the efficacy of cisplatin-based chemotherapy in non-small-cell lung cancer (NSCLC). Although several ongoing trials are evaluating the level of expression of ERCC1, no consensus has been reached regarding a method for evaluation. METHODS: We used the 8F1 antibody to measure the level of expression of ERCC1 protein by means of immunohistochemical analysis in a validation set of samples obtained from 494 patients in two independent phase 3 trials (the National Cancer Institute of Canada Clinical Trials Group JBR.10 and the Cancer and Leukemia Group B 9633 trial from the Lung Adjuvant Cisplatin Evaluation Biology project). We compared the results of repeated staining of the entire original set of samples obtained from 589 patients in the International Adjuvant Lung Cancer Trial Biology study, which had led to the initial correlation between the absence of ERCC1 expression and platinum response, with our previous results in the same tumors. We mapped the epitope recognized by 16 commercially available ERCC1 antibodies and investigated the capacity of the different ERCC1 isoforms to repair platinum-induced DNA damage. RESULTS: We were unable to validate the predictive effect of immunostaining for ERCC1 protein. The discordance in the results of staining for ERCC1 suggested a change in the performance of the 8F1 antibody since 2006. We found that none of the 16 antibodies could distinguish among the four ERCC1 protein isoforms, whereas only one isoform produced a protein that had full capacities for nucleotide excision repair and cisplatin resistance. CONCLUSIONS: Immunohistochemical analysis with the use of currently available ERCC1 antibodies did not specifically detect the unique functional ERCC1 isoform. As a result, its usefulness in guiding therapeutic decision making is limited. (Funded by Eli Lilly and others.).

PARP1 impact on DNA repair of platinum adducts: preclinical and clinical read-outs.

Evaluation of DNA repair proteins might provide meaningful information in relation to prognosis and chemotherapy efficacy in Non-Small Cell Lung Cancer (NSCLC) patients. The role of Poly(ADP-Ribose) Polymerase (PARP) in DNA repair of platinum adducts has not been firmly established. We used a DNA repair functional test based on antibody recognition of cisplatin intrastrand platinum adducts on DNA. We evaluated the effect of PARP inhibition on DNA repair functionality in a panel of cisplatin cell lines treated by the clinical-grade pharmacological inhibitor CEP8983 (a 4-methoxy-carbazole derivate) and the commercially available inhibitor PJ34 (phenanthridinone). We determined PARP1 protein expression in whole tumor sections from the International Adjuvant Lung cancer Trial (IALT)-bio study and tested a 3-marker PARP1/MSH2/ERCC1 algorithm combining PARP1 tumor status with previously published data. Chemosensitivity of cisplatin in NSCLC cell lines was correlated with the accumulation of cisplatin DNA adducts (P=0.0004). Further, the pharmacological inhibition of PARP induced a 1.7 to 2.3-fold increase in platinum adduct accumulation (24h) in A549 cell line suggesting a slow-down of platinum DNA-adduct repair capacity. In parallel, PARP1 inhibition increased the sensitivity to cisplatin treatment. In patient samples, PARP1 expression levels did not influence patient survival or the effect of platinum-based post-operative chemotherapy in the global IALT-bio population (interaction P=0.79). Among cases with high expression of all three markers (triple positive), untreated patients had prolonged survival with a median DFS of 7.8 years, (HR=0.34, 95%CI [0.19-0.61], adjusted P=0.0003) compared to triple negative patients (1.4 years). Remarkably, triple positive patients suffered from a detrimental effect (4.9-year reduction of median DFS) by post-operative cisplatin-based chemotherapy (HR=1.79, 95%CI [1.01-3.17], adjusted P=0.04, chemotherapy vs. control). Combinatorial sub-group analysis of the 3 markers further suggested that PARP1 tumor positivity might constitute a molecular context with high theranostic interest of ERCC1 and MSH2 in NSCLC. In conclusion, our data confirm that platinum DNA adduct accumulation is linked to chemosensitivity, which increase by pharmacological PARP inhibitors points to a role of PARP-dependent DNA repair in the process. We further suggest DNA repair biomarkers should be analyzed in a larger context of multiple DNA repair pathway regulation.

19q13-ERCC1 gene copy number increase in non--small-cell lung cancer.

BACKGROUND: Excision repair cross complementing 1 gene expression level has potential as a prognostic and predictive marker of the efficacy of chemotherapy in NSCLC. The effect of ERCC1 gene copy number (CN) variation (CNV) on ERCC1 expression and the clinical outcome of patients with NSCLC are not known. MATERIALS AND METHODS: Copy number variation of the 19q13.3 region carrying the ERCC1 gene, classified as gene amplification (GA) or high polysomy (HP), was evaluated on 235 formalin-fixed and paraffin-embedded tumors from resected NSCLC patient samples and 16 NSCLC cell lines using FISH. We analyzed the potential correlations between FISH status and ERCC1 expression, patient's outcome, and cisplatin sensitivity in the cohort or cell lines. RESULTS: An increase of 19q13.3 gene CN was detected in 60 cases (25.5%) including 27 cases with GA and 33 cases with HP. A nonsignificant trend for higher ERCC1 expression in HP patients compared with GA and patients with low CNV was found (P = .06). In patients not treated with chemotherapy, FISH negative status cases had longer disease-free survival (DFS) compared with patients with 19q13-ERCC1 GA (P = .02). A 3-fold increase in IC50 of cisplatin in cell lines with high 19q13-ERCC1 CN compared with cells without CNV was shown. CONCLUSION: ERCC1 CN increase assessed using FISH did not determine ERCC1 expression status but yields potential prognostic information on DFS in untreated patients with NSCLC. The clinical relevance of an association of 19q13-ERCC1 FISH status and chemosensitivity or prognosis in patients needs further investigation and validation.

The potential of exploiting DNA-repair defects for optimizing lung cancer treatment.

The tumor genome is commonly aberrant as a consequence of mutagenic insult and incomplete DNA repair. DNA repair as a therapeutic target has recently received considerable attention owing to the promise of drugs that target tumor-specific DNA-repair enzymes and potentiate conventional cytotoxic therapy through mechanism-based approaches, such as synthetic lethality. Treatment for non-small-cell lung cancer (NSCLC) consists mainly of platinum-based chemotherapy regimens and improvements are urgently needed. Optimizing treatment according to tumor status for DNA-repair biomarkers, such as ERCC1, BRCA1 or RRM1, could predict response to platinum, taxanes and gemcitabine-based therapies, respectively, and might improve substantially the response of individual patients' tumors. Finally, recent data on germline variation in DNA-repair genes may also be informative. Here, we discuss how a molecular and functional DNA-repair classification of NSCLC may aid clinical decision making and improve patient outcome.

Brain-derived neurotrophic factor and neurotrophin-4/5 are expressed in breast cancer and can be targeted to inhibit tumor cell survival.

PURPOSE: Given that nerve growth factor has previously been shown to be involved in breast cancer progression, we have tested here the hypothesis that the other neurotrophins (NT) are expressed and have an influence in breast tumor growth. EXPERIMENTAL DESIGN: The expression of brain-derived neurotrophic factor (BDNF), NT-3 and NT-4/5, as well as the neurotrophin receptor p75(NTR), TrkB, and TrkC, was studied by RT-PCR, Western blotting, and immunohistochemistry in cell lines and tumor biopsies. The biological impacts of neurotrophins, and associated mechanisms, were analyzed in cell cultures and xenografted mice. RESULTS: BDNF and NT-4/5 were expressed and secreted by breast cancer cells, and the use of blocking antibodies suggested an autocrine loop mediating cell resistance to apoptosis. The corresponding tyrosine kinase receptor TrkB was only rarely observed at full length, whereas the expression of TrkB-T1, lacking the kinase domain, as well as p75(NTR), were detected in all tested breast cancer cell lines and tumor biopsies. In contrast, NT-3 and TrkC were not detected. SiRNA against p75(NTR) and TrkB-T1 abolished the antiapoptotic effect of BDNF and NT-4/5, whereas the pharmacological inhibitors K252a and PD98059 had no effect, suggesting the involvement of p75(NTR) and TrkB-T1, but not kinase activities from Trks and MAPK. In xenografted mice, anti-BDNF, anti-NT-4/5, anti-p75(NTR), or anti-TrkB-T1 treatments resulted in tumor growth inhibition, characterized by an increase in cell apoptosis, but with no change in proliferation. CONCLUSION: BDNF and NT-4/5 contribute to breast cancer cell survival and can serve as prospective targets in attempts to inhibit tumor growth.

A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.

Inhibition of tumor necrosis factor-induced phenotypes by short intracellular versions of latent membrane protein-1.

Tumor necrosis factor (TNF) is a potent multi-functional cytokine with a homeostatic role in host defence. In case of deregulation, TNF is implicated in numerous pathologies. The latent membrane protein-1 (LMP1) is expressed by Epstein-Barr virus during viral latency and displaying properties of a constitutively activated member of the TNF receptor family. Both TNFR1 and LMP1 share a similar set of proximal adapters and signalling pathways although they display different biological responses. We previously demonstrated that the intracellular part of LMP1, LMP1-CT, a dominant-negative form of LMP1, inhibits LMP1 signalling. Here, we developed shorter versions derived from C-terminal part of LMP1 to investigate their roles on LMP1 and TNF signalling. We constructed several mutants of LMP1 containing a part of cytoplasmic signalling region fused to the green fluorescent protein. These mutants selectively impair signalling by LMP1 and TNF but not by IL-1beta which uses other adapters. Dominant-negative effect was due to binding and sequestration of LMP1 adapters RIP, TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. Expression of these mutants impairs the recruitment of these adapters by TNFR1 and TNF-associated phenotypes. These mutants did not display cytostatic properties but were able to modulate TNF-induced phenotypes, apoptosis or cell survival, depending on the cell context. Interestingly, these mutants are able to inhibit a pro-inflammatory response in endothelial cells. These data demonstrate that LMP1 derived molecules can be used to design compounds with potential therapeutic roles in diseases due to TNF overactivation.

Nerve growth factor is a potential therapeutic target in breast cancer.

We show here that nerve growth factor (NGF), the prototypic neurotrophin, can be targeted in breast cancer to inhibit tumor cell proliferation, survival, and metastasis. Analysis of a series of biopsies revealed widespread expression of NGF in the majority of human breast tumors, with anti-NGF immunoreactivity concentrated in the epithelial cancer cells. Moreover, immunodeficient mice xenografted with human breast cancer cells and treated with either anti-NGF antibodies or small interfering RNA against NGF displayed inhibited tumor growth and metastasis. Such treatments directed against NGF induced a decrease in cell proliferation with a concomitant increase in apoptosis of breast cancer cells and an inhibition of tumor angiogenesis. Together, these data indicate that targeting NGF in breast cancer may have therapeutic ramifications.