bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A34: Nitric Oxide enhances inflammation, microRNA-21 expression and KRAS-induced lung carcinogenesis
Authors: Okayama Hirokazu , Saito Motonobu , Naohide Oue , Weiss Jonathan M. , Stauffer Jimmy , Takanoshita Seiichi , Wiltrout Robert H. , Hussain S. Perwez , Harris Curtis C.
Published: March 2013

Mutant KRAS in lung cancers induce molecular pathways that regulate cellular proliferation, survival and inflammation, which enhance tumorigenesis. Inducible nitric oxide synthese (NOS2) up-regulation and sustained nitric oxide (NO•) generation are induced during the inflammatory response and correlate positively with lung tumorigenesis. To explore the mechanistic contribution of NOS2 to KRAS-induced lung tumorigenesis and inflammation, we used a genetic strategy of crossing NOS2 knockout (NOS2KO) C57BL6 inbred mice with a KRASG12D-driven mouse lung cancer model. KRASG12D/NOS2KO mice exhibited delayed lung tumorigenesis, resulting in a longer overall survival time compared with that of KRASG12D/NOS2WT (wildtype) controls. Correspondingly, tumors in KRASG12D/NOS2KO mice had reduced tumor cell proliferation in adenomas and carcinomas. NOS2-deficiency also led to dramatically suppressed inflammatory response by attenuation of macrophage recruitment into alveoli and within tumor foci. In contrast, FOXP3+ regulatory T cells were increased in tumors from KRASG12D/NOS2KO mice. We further analyzed the expression of microRNA-21 (miR-21), an oncogenic non-coding RNA involved in oncogenic Ras signaling, by quantitative reverse transcription PCR and in situ hybridization. Lung carcinomas dissected from KRASG12D/NOS2KO mice showed a significantly reduced miR-21 expression along with decreased tumor cell proliferation, suggesting that NOS2-deficiency could attenuate RAS signaling pathways that transactivate miR-21 expression. Therefore, deletion of NOS2 decreases lung tumor growth as well as inflammatory responses initiated by oncogenic KRAS, suggesting that both KRAS and NOS2 cooperate in driving lung tumorigenesis and inflammation. Inhibition of NOS2 may have a therapeutic value in lung cancers with oncogenic KRAS mutations.
Abstract A35: The role of Mdm2 cleavage in p53 function and chemotherapy response
Authors: Oliver Trudy G.
Published: March 2013

Chemotherapy resistance is a major obstacle in cancer treatment, yet the mechanisms of resistance to specific therapies have been largely unexplored in vivo. Employing genetic, genomic, and imaging approaches, we have examined the dynamics of response to a mainstay chemotherapeutic, cisplatin, in multiple mouse models of human non-small cell lung cancer (NSCLC). We show that lung tumors initially respond to cisplatin and exhibit a significant reduction in tumor burden. Prolonged cisplatin treatment promotes the emergence of resistant tumors with enhanced repair capacity that are cross-resistant to platinum analogs, exhibit advanced histopathology, and possess an increased frequency of genomic alterations. Cisplatin-resistant tumors express elevated levels of multiple DNA damage repair and cell cycle arrest-related genes, including p53-inducible protein with a death domain (Pidd).

We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a novel cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress. Our current goals are to: 1) elucidate how cleaved Mdm2 dictates p53 function and 2) create mouse models to manipulate Mdm2 cleavage in vivo in order to interrogate the role of Mdm2 cleavage in lung tumorigenesis and drug response.
Abstract A44: K-homology splicing regulatory protein modulates tumor suppressor miRNA 29b through the noncanonical Wnt 7a signaling pathway
Authors: Wilson Lora A. , Winn Robert A.
Published: March 2013

Precedent has established the role of the Wnt/β-catenin pathway in oncogenesis. Several members of the Wnt family are known to be tumor promoting in non-small cell lung cancer, NSCLC. In this pathway β-catenin, a downstream component, accumulates and serves as a T-cell transcription/lymphoid enhancer factor. We previously identified the Wnt ligand in the lung, Wnt 7a, as a member of the non-canonical pathway/β-catenin independent pathway, which inhibits transformed cell growth. K-homology splicing regulatory protein (KSRP) is a member of the family of AU rich element binding proteins. It possesses four K-homology motifs and causes decay of specific mRNA transcripts and regulation of miRNA. Wnt 7a positively regulates the expression of KSRP. As a result, KSRP upregulates the processing of miRNA 29b. The miRNA 29b family is comprised of 29a, 29b, and 29c. KSRP and miRNA 29b expression are elevated in nontransformed cells and alternatively, lower levels of expression are in NSCLC. The role of KSRP regulating miRNA 29b in the non-canonical Wnt pathway in the lung has previously been unknown. Over expression of KSRP through upregulation of miRNA 29b in NSCLC cells decreases cell proliferation in MTS assays, a measure of proliferation, as well as reducing colony formation in clonogenic assays. We reveal a novel association of p53 and miRNA 29b. We identified that KSRP increases PPARγ activation which in turn positively modulates miRNA 29b, resulting in upregulation of p53. The antitumorigenic effect is specific to Wnt 7a activation through Erk 5 dependent kinases.
Abstract B17: The small RhoGTPase RhoB as a new potential marker of EGFR-TKI resistance
Authors: Marty Claire , Favre Gilles , Mazieres Julien
Published: March 2013

Introduction: EGFR tyrosine kinase inhibitors (EGFR-TKI) are widely used in metastatic non small cell lung cancer (NSCLC). Nevertheless, the majority of patients with wild type EGFR do not respond to these treatments and, in the selected group with EGFR mutations, we some primary resistance and all the patients will ultimately develop acquired resistance.

The small RHO GTPase RhoB is involved in cell proliferation, invasion, apoptosis and can act as a tumor suppressor gene in lung carcinogenesis by controlling survival and invasion through AKT pathway. Furthermore, RhoB participates in the regulation of endocytic trafficking of EGFR. Specifically, activated RhoB is able to delay the transport of internalized EGFR to lysosomes, thus modifying its downstream signaling.

The objective of our study is to know if RhoB is a molecular determinant of the response to the EGFR-TKI erlotinib, and if RhoB could be a marker for erlotinib resistance.

Material and Method: We used various lung cancer cell lines harboring either wild-type or mautated EGFR and K-Ras. RhoB was either overexpressed by adenovirus transfection or inhibited by interfering RNA (siRNA) in these cells. Cell proliferation after 72h of treatment with erlotinib was measured by a colorimetric assay. In the same way, we performed invasion assays and western-blot analyses of downtsream signaling pathways.

Results: (1) RhoB overexpression decreases the sensitivity to erlotinib of cells presenting a wild type or mutated EGFR in a dose-dependent manner whereas the sensitivity to erlotinib of cells bearing KRas mutation was not affected by RhoB overexpression. (2) Erlotinib-induced inhibition of cellular invasion is impaired by overexpression of RhoB in wild type or mutated EGFR cell lines. (3) The phosphorylation of Akt (and not of ERK) is inhibited by erlotinib and is restored upon overexpression of RhoB invasion.

Conclusions: Our results suggest that RhoB is involved in the sensitivity to erlotinib in wild-type K-Ras cells harboring either mutated or wild-type EGFR. RhoB is able to block erlotinib-induced inhibition of cell proliferation and invasion mainly through AKT pathway modulation. We plan to extend our in vitro findings in a cohort of patients treated with erlotinib.
Abstract B39: Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer
Authors: Watanabe Kousuke , Goto Akiteru , Fukayama Masashi , Nagase Takahide , Yatomi Yutaka , Ohishi Nobuya , Takai Daiya , Emoto Noriko , Hamano Emi , Amano Yosuke , Ishikawa Rie , Sunohara Mitsuhiro , Kawakami Masanori , Kitano Kentaro , Nakajima Jun
Published: March 2013

microRNA expression is frequently altered in human cancers. Epigenetic silencing, especially DNA methylation, is one of many mechanisms of microRNA suppression in cancer. To search for epigenetically silenced microRNAs in non-small-cell lung cancer (NSCLC), we mapped human microRNAs on autosomal chromosomes and selected in silico 55 microRNAs that met one of the following criteria: (i) microRNAs within CpG islands, (ii) microRNAs within 1 kbp downstream of CpG islands, and (iii) microRNAs within gene introns whose promoters have CpG islands. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 microRNAs. Fourteen microRNAs (mir-375, mir-196b, mir-126, mir-34b, mir-127, mir-203, mir-148a, mir-181c, mir-30e, mir-449a, mir-340, mir-486, mir-483, mir-139) were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. Among the 14 microRNAs, seven (mir-126, mir-34b, mir-203, mir-30e, mir-449a, mir-486, mir-139) were frequently suppressed in primary NSCLCs. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, while mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. A significant enrichment of H3K27me3 was observed in lung cancer cell lines with decreased mir-126 expression, suggesting the involvement of a polycomb complex in the regulation of EGFL7 and mir-126. The overexpression of mir-34b and mir-126 by plasmid vectors decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these microRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41% and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.
Abstract B4: A “DNA replication stress” gene signature associated with a poor prognosis in early non-small cell lung cancer
Authors: Allera-Moreau Camille , Delisle Marie-Bernadette , Hoffmann Jean-Sebastien , Cazaux Christophe , Rouquette Isabelle , Lepage Benoit , Oumouhou Naima , Gordien Karine , Brouchet Laurent , Mazieres Julien , Pasero Philippe
Published: March 2013

The conventional clinical staging classification is not sufficient to predict the survival of patients who suffer from early lung cancer. Additional prognostic factors are then needed to better forecast their outcome.

Since perturbation of the genome replication i.e. the so-called “replicative stress” is admitted to contribute to neoplasia from its early stages, we hypothesized that genes involved in such process may therefore represent a still under-explored source of such biomarkers.

We specifically assessed in primary tumors and adjacent normal tissues from a series of 93 patients suffering from early- or mid-stage non-small cell lung adenocarcinoma the expression (RT-qPCR) of 77 “DNA replication” genes involved in either initiation/licensing at the 50,000 replication origins dispersed along the genome, elongation of DNA chains onto damaged or undamaged DNA, maintenance of stalled DNA replication forks or intra-S phase DNA damage processing and signaling.

A 4-gene signature separated patients to high-risk and low-risk subgroups with significantly different survival. This prognostic effect was independent on age, sex, treatment, stage classification and expression of proliferation markers.

We propose that a “cancer replisome” signature could be a predictor of the cancer survival and might also help understanding the molecular mechanisms underlying tumor progression in lung cancer patients.
Abstract B41: Consider the source: Normal lung expression classifies cancer histologies, subtypes, and mutations
Authors: Wilkerson Matthew D. , Randell Scott H. , Hayes D. Neil
Published: March 2013

Lung cancer's pathogenesis from normal cell to metastatic disease involves vast and complex genetic alterations. However, a patient's genetic alterations do not completely explain the pathogenic process, for tumors with the same alterations have different features and therapy responses. We hypothesized that differences in ancestral anatomical regions affect pathogenesis and as such would correlate with differences in cancer histology, subtype, and mutations. Anatomical distal and proximal region specimens from normal lungs were collected and assayed by Agilent 44K gene expression microarrays and immunohistochemistry (IHC). 271 surgically extracted lung cancers were assayed by gene expression, IHC, and gene sequencing for EGFR, KRAS, STK11, and TP53. Using the normal lung specimens, a novel classifier was created to assign cancers with an anatomical region using gene expression alone. Cancers classified as distal by gene expression were 84% adenocarcinoma, while 73% of those classified as proximal were squamous cell carcinoma (P<0.001). While this trend of histology and region is consistent with conventional wisdom, our results also suggest that lung cancers retain substantial gene expression from their ancestral anatomical region. Interestingly, the remainder of the cancers that did not follow this trend was explained by our previously described molecular subtypes (1, 2). Of adenocarcinomas classified proximal, 92% were in the Magnoid molecular subtype. Of squamous cell carcinomas classified distal, 60% were in the Secretory molecular subtype. Complementing microarray gene expression, TTF1 IHC protein expression was greater in distal normal specimens compared to proximal. Consistent with distal classification, adenocarcinomas had the greatest TTF1 expression. Of the squamous molecular subtypes, the Secretory subtype exhibited the greatest TTF1 expression, consistent with its distal classification. This Secretory subtype may explain previously reported TTF1-positive squamous cell carcinomas. Finally, gene mutation rates were significantly different between cancers classified as proximal and as distal. EGFR and KRAS mutation rates were greater in distal tumors compared to proximal tumors. In contrast, TP53 mutation rates were greater in proximally classified tumors. These results suggest that ancestral anatomical regions may affect lung cancer pathogenesis by modifying susceptibility to and dependency on different gene mutations. In summary, normal lung anatomical gene expression classifies lung cancers with different histologies, molecular subtypes, and gene mutation rates.
Abstract B44: Development of a serum test for the detection of lung cancer based on oncogenic BARD1 isoform expression
Authors: Irminger-Finger Irmgard , André Pierre-Alain , Pilyugin Maxim , Hegedus Balazs , Laurent Geoffrey J. , Bianco Andrea
Published: March 2013

The BARD1 protein was originally identified as binding partner of the breast cancer gene product, BRCA1. Highly upregulated expression of aberrant isoforms of BARD1, derived from differential splicing, was correlated with poor prognostic factors in breast and ovarian cancer (Wu et al. Int J Can, 2006; Li et al. Can Res, 2007) and decreased patient survival in lung cancer (Zhang et al. Int J Can, 2011).

Previous and ongoing studies have shown that BARD1 isoforms act antagonistically to the functions of BARD1 and BRCA1 as ubiquitin ligase. In particular, BARD1beta is promoting cell proliferation by stabilizing the Aurora kinases (Ryser et al. Can Res 2009; Bosse et al. submitted). Isoforms BARD1-beta and BARD1-pi are specifically upregulated in lung cancer and correlated with poor prognosis (Zhang et al., Int J Can, 2011). Thus, BARD1 isoforms might be drivers of tumorigenesis and potential markers of lung cancer progression, if detectable in the patients' sera.

To this goal, we performed ELISA tests with antibodies against different regions of BARD1 for the detection of BARD1 isoforms in the blood of lung cancer patients. We also generated a peptide library representing 40 epitopes mimicking BARD1 isoforms, for the detection of autoimmune antibodies recognizing epitopes expressed by BARD1 isoforms.

BARD1 protein isoforms could be detected by ELISA in various serum samples, however this detection was always reproducible due to the BARD1 protein instability. We then performed inverse ELISA assays, using peptides for capturing autoimmune antibodies directed against BARD1 isoforms. Using serum samples from 60 non-small cell lung cancer (NSCLC) from time of diagnosis, and 40 control sera from phenotypically healthy volunteers, we could distinguish NSCLC cancer patients and controls. When applying a combination of seven peptides lung cancer was detected with 87 percent sensitivity and 68 percent specificity.

Thus, our data show convincingly that antibodies against BARD1 isoforms are telltales of lung cancer and their detection can be further developed towards a blood test for the early detection of lung cancer. Experiments including larger patients and control group numbers, sera from patients with different types of lung cancer, as well as the comparison of this BARD1 isoform test with the standard test for lung cancer detection (CT scan), are currently ongoing, and should lead to optimized test conditions and a definition of the target patient set.
Abstract B46: Clinicopathologic implications of ALK rearrangement in surgically resected NSCLC: A proposal of molecular algorithm with a small lung biopsy of adenocarcinoma
Authors: Ho Paik Jin , Choi Chang-Min , Kim Hyojin , Jang Se Jin , Sung Sook-Whan , Kim Dong Kwan , Kim Hwa Jung , Yoon Hoil , Lee Choon-Taek , Jheon Sanghoon , Choea Ji-Young , Chung Jin-Haeng
Published: March 2013

Background: To characterize the clinicopathologic features of ALK-rearranged lung cancer, and suggest a molecular test protocol for lung adenocarcinoma in the small biopsy specimen.

Methods: In 735 NSCLC surgical specimens, clinicopathologic features, ALK protein over-expression by immunohistochemistry (IHC), and ALK rearrangement by fluorescence in situ hybridization (FISH) as well as EGFR and KRAS mutation studies were analyzed.

Results: Of the 735 NSCLC cases, 28 (3.8%) were ALK FISH-positive. ALK rearrangement, EGFR and KRAS mutation were mutually exclusive. ALK rearrangement was significantly higher in adenocarcinomas (6.8%, p<0.001), younger age (p<0.0007), women (7.6%, p<0.001), and never-smokers (8.9%, p<0.001) with no gender difference in the adenocarcinoma or never-smoker subgroup. ALK FISH-positivity was not associated with disease recurrence (HR, 0.79; 95% CI, 0.42–1.49) or overall survival (HR, 0.61; 95% CI, 0.24–1.55). However, ALK-rearranged lung cancer tended to show more frequent lymph node metastasis despite its lower T stage. Similar to EGFR-mutated lung cancer, ALK-rearranged lung cancer was enriched in adenocarcinoma, women, and never-smokers. The results of ALK IHC and FISH obtained from tissue microarray (TMA)/biopsy specimens and whole sections after resection were concordant.

Conclusion: ALK rearrangement was not a significant prognostic factor in surgically resectable NSCLC. The clinical profiles of ALK-rearranged lung cancer patients overlapped with those of EGFR-mutated patients. Therefore, we suggest that simultaneous tests for ALK IHC and EGFR mutation (Chung's SNUBH molecular test protocol), which has important implications for the storage and use of small biopsy or cytology samples for genetic analysis.
Abstract B6: Folylpoly-glutamate synthetase and thymidylate synthase are associated with clinical outcome from pemetrexed-based therapy in non-small cell lung cancer (NSCLC)
Authors: Christoph Daniel C. , Loewendick Heike , Peglow Anja , Schuler Martin , Eberhardt Wilfried E. , Hirsch Fred R. , Asuncion Bernadette Reyna , Wynes Murry W. , Gauler Thomas C. , Wohlschlaeger Jeremias , Theegarten Dirk , Welter Stefan , Tran Cindy , Hassan Biftu
Published: March 2013

Background: The antifolate pemetrexed targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, pemetrexed is converted to polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict outcome following pemetrexed-treatment of patients with advanced NSCLC, like in malignant pleural mesotheliomas (Christoph et al., J Clin Oncol. 2011: 29 suppl.). This is the largest report on pemetrexed-treatment outcome based on TS and FPGS in Caucasian patients with advanced NSCLC.

Methods: Pretreatment tumor samples from 161 patients (pts) with metastatic NSCLC, treated with pemetrexed combined with platinum (74 pts (46%)) or as single agent (82 pts (51%)) or within other combinations (5 pts (3%)), were retrospectively analyzed. FPGS and TS protein expression levels were evaluated by IHC using the H-Scoring system (0–300), which relies on the product of intensity (range 0 to 3) and the percentage of positive tumor cells (0–100%). Radiographic evaluation of response was performed according to RECIST criteria (version 1.1).

Results: Median pretreatment H-scores were 180 for FPGS (range: 0–280) and 205 (range: 120–290) for TS. Using the log-rank test and the median H-score as cut-off, we found a significant association between low TS protein expression and improved progression-free survival (PFS) (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.66, 95% CI, 0.45 to 0.96; P=0.03) or prolonged overall survival (median OS of 33.9 months vs. 15.0 months; hazard ratio [HR] 0.52, 95% CI, 0.31 to 0.86; P=0.01). Moreover, high FPGS protein expression was only associated with better PFS (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.58, 95% CI, 0.37 to 0.89; P=0.03). Considering exclusively patients suffering from adenocarcinomas (110 pts (68%)), TS was associated with objective response to pemetrexed-based treatment (mean H-score 192 for responders vs. 210 for non-responders, P=0.03).

Conclusions: We have investigated FPGS and TS protein expressions in tumor specimens from the largest series of pemetrexed-treated Caucasian NSCLC patients. Baseline determination of TS and FPGS expression by IHC using the H-score system is associated with clinical outcome from pemetrexed-based therapy in advanced NSCLC. Further prospective validation studies are warranted.
Abstract A14: Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer
Authors: Ward Aoife , Balwierz Aleksandra , Zhang Jitao David , Kueblbeck Moritz , Wiemann Stefan , Hielscher Thomas , Pawitan Yudi , Sahin Ozgur
Published: March 2013

Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis. It has been shown to occur in resistance to a range of cancer therapies, including tamoxifen. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. To investigate the role of miRNAs in the development of resistance to tamoxifen as well as accompanying EMT-like properties, we established a tamoxifen resistant (TamR) model by continually exposing MCF-7 breast cancer cells to tamoxifen. In addition to the molecular changes known to be involved in acquired tamoxifen resistance, TamR cells displayed mesenchymal features and had increased invasiveness. Genome-wide miRNA microarray analysis revealed that miRNA-375 was among the top down-regulated miRNAs in resistant cells. Re-expression of miR-375 was sufficient to sensitize TamR cells to tamoxifen and partly reversed EMT. A combination of mRNA profiling, bioinformatic analysis, and experimental validation identified metadherin/MTDH as a direct target of miR-375. Knockdown of MTDH partially phenocopied the effects of miR-375 on the sensitivity to tamoxifen and the reversal of EMT. We observed an inverse correlation between the expression of miR-375 and its target MTDH in primary breast cancer samples, implying the pathological relevance of targeting. Finally, tamoxifen-treated patients with higher expression of MTDH had a shorter disease-free survival and higher risk of relapse. As most cancer-related deaths occur due to resistance to standard therapies and metastasis, re-expression of miR-375 or targeting MTDH might serve as potential therapeutic approaches for the treatment of tamoxifen resistant breast cancer.
Abstract A16: Molecular mechanisms of EGFR therapy resistance in breast cancer cells
Authors: Castillo-Pichardo Linette , Borrero-Garcia Luis D. , Redis Roxana S. , Calin George A. , Cubano Luis A. , Dharmawardhane Suranganie
Published: March 2013

The purpose of this study is to investigate molecular mechanisms of epidermal growth factor receptor (EGFR) therapy resistance using a syngeneic pair of gefitinib-sensitive and gefitinib-resistant breast cancer cells. Signaling from EGFRs is usually dysregulated in breast, as well as other cancers, where it is commonly associated with an aggressive phenotype and poor outcome. The initial success of targeted therapies for EGFR1 and HER2 has been marred by de novo or acquired resistance. Gefitinib, a small molecule tyrosine kinase inhibitor with high specificity towards EGFR, was proven to be effective for the treatment of non-small cell lung carcinoma (NSCLC). However, clinical trials of gefitinib as a breast cancer therapeutic have failed due to lack of response, probably due to resistance, and adverse toxic effects. We have developed a Gefitinib resistant variant of the gefitinib sensitive human breast cancer cell line SKBR3 (SKBR3 G.R.), with the objective of studying the involvement of micro (mi)-RNAs in therapy resistance. A partial screening of several miRNAs previously shown to be involved in therapy resistance was performed and miRNA (miR-)200a was identified as a downregulated miRNA in the SKBR3 G.R. cells. Members of the miR-200 family play a critical role in the suppression of epithelial-to-mesenchymal transition (EMT) by repressing key mRNAs involved in this process. Additionally, the miR-200 family can regulate tumor cell adhesion, migration, invasion and metastasis; and has been associated with EGFR inhibitor resistance. Herein, we have analyzed the protein expression of EGFR, phospho-EGFR, and miR-200 family targets Zeb1, β-catenin, and the ErbB receptor inhibitor-1 (ERRFI-1 or Mig6) by western blot analysis in SKBR3 and SKBR3 G.R. breast cancer cells. Our results show a significant increase in the expression of EGFR, Zeb1, and β-catenin; whereas, phospho-EGFR and Mig6 expression was significantly downregulated in SKBR3 G.R. cells when compared to parental cells. Therefore, Mig6 expression may not be regulated via miR-200 activity in these cells. Downregulation of Mig6 may explain the increased EGFR levels due to its role in EGFR internalization and subsequent degradation. Since SKBR3 cells are mesenchymal, they do not express E-cadherin, another Zeb1 target. Our results suggest that in cancer cells that have undergone EMT, decreased miR-200a levels, and thus, increased Zeb1 and β-catenin, a potent pro-cancer transcriptional activator in the absence of an E-cadherin axis, is a potential mechanism of Gefitinib resistance in aggressive breast cancer.

This study was supported by NIH/NIGMS SC3GM094824 to SD, the UCC Pilot Project Program, and the UCC Postdoctoral Program sponsored by the PR Science, Technology and Research Trust.
Abstract A18: Reduced NF1 expression in resistance to EGF receptor inhibitory drugs in lung adenocarcinoma
Authors: de Bruin Elza , Cowell Catherine , Howell Michael , Varmus Harold , Politi Katerina , Downward Julian
Published: March 2013

Tyrosine kinase inhibitors, such as erlotinib, exhibit striking efficacy in lung adenocarcinomas with activating mutations in the epidermal growth factor receptor (EGFR). However, patients inevitably develop therapeutic resistance, often due to second site mutations (T790M) in the EGFR. In this study, we aim to identify novel determinants of resistance in order to find new drug targets that may increase the effectiveness of EGFR targeted therapy.

We performed a genome-wide siRNA screen using the PC9 cell line, which harbors an EGFR exon 19 deletion mutation and is exquisitely sensitive to erlotinib. From this screen, we identified genes that upon knockdown had a significantly different effect on cell survival in drug-treated versus untreated conditions. Within the list of candidate genes is the tumor suppressor NF1, encoding the RAS GTPase-activating protein neurofibromin. In addition, low Nf1 expression is associated with the acquisition of resistance in a preclinical mouse model of activated mutant EGFR-driven lung cancer in tumors where the second site mutation in EGFR did not occur.

In vitro experiments show that decreased expression of NF1 results in a failure of erlotinib to fully inhibit RAS-ERK signaling. Treatment of NF1-deficient lung cancer cells with a MEK inhibitor restored their sensitivity to erlotinib both in in vitro and in xenograft experiments. Interestingly, approximately half of the erlotinib resistant tumors in our mouse model regressed upon treatment with erlotinib in combination with a MEK inhibitor, while tumors treated with erlotinib only continued to progress. This suggests that combination of a MEK inhibitor and an EGFR inhibitor may be an effective approach to the treatment of some cases of drug resistant lung cancer.
Abstract A3: mTOR signaling and tamoxifen response in postmenopausal breast cancer
Authors: Bostner Josefine , Karlsson Elin , Pandiyan Muneeswaran Jayachandra , Fornander Tommy , Nordenskjöld Bo
Published: March 2013

mTOR is a central, frequently altered, node for the complex growth signaling in breast cancer. Activation of the PI3K/Akt/mTOR-pathway seems to be one of the mechanisms for patients recurring after adjuvant endocrine treatment. Growth factor signals promote phosphorylation of the estrogen receptor alpha (ER), thereby altering the receptor conformation, stability and its response to tamoxifen treatment. Ongoing studies are investigating the possibility to inhibit the mTOR pathway in addition to endocrine treatment or after endocrine treatment failure. In the present retrospective study, a postmenopausal early breast cancer cohort of 812 patients, randomized to tamoxifen or no endocrine treatment, was analyzed according to the protein expression of mTOR phosphorylated on serine 2448. Downstream targets of mTOR are the S6Kinases, S6K1 and S6K2. S6K1 is thought to directly phosphorylate the ER on serine 167. Here, a positive correlation between p-mTOR and p-ERs167 was shown. However, no correlation was detected between p-mTOR and p-ERs305, indicating that the active mTOR signaling specifically targets the ERs167 residue. Activated 4EBP1, another common mTOR target, did not correlate with the p-mTOR protein expression. No prognostic value, independently of cut-off, was seen for p-mTOR expression in this material.

Analyses of p-mTOR as a tool for treatment prediction showed that negative, low and medium expression in the tumor did not affect the patients response to tamoxifen, p=0.0001. High and abundant expression decreased the tamoxifen sensitivity to a non-significant degree, p=0.34. An ER-positive/PgR-positive selection further improved the hypothesis of p-mTOR being an indicator of tamoxifen resistance. Where the p-mTOR negative group responded well to treatment (p<0.00001), compared with the p-mTOR positive group, where the treated group did not separate significantly from the controls (p=0.71).

In conclusion, the results indicate that risk of recurrence after tamoxifen treatment may increase in patients with high mTOR kinase activity.
Abstract A45: Signaling Events Associated with Cytoprotective Induction of Nitric Oxide Synthase in a Photodynamic Therapy Model
Authors: Bhowmick Reshma , Girotti Albert W.
Published: March 2013

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and photosensitizer-exciting light to produce reactive oxygen species that lead to tumor eradication. Nitric oxide (NO) produced by tumor or tumor vasculature cells could be pro-carcinogenic by inhibiting apoptosis and/or promoting angiogenesis and tumor growth. We recently showed that photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX in mitochondria of COH-BR1 breast tumor cells strongly upregulated inducible nitric oxide synthase (iNOS) and steady state NO level in these cells. Including the iNOS inhibitor 1400W during photochallenge dramatically enhanced caspase-9 activation and Annexin-assessed apoptotic cell killing while reducing NO production assessed by the intracellular NO probe DAF-2DA. Short hairpin RNA (ShRNA)-based iNOS knockdown (kd) produced similar results, confirming iNOS involvement. An apoptosis-promoting effect of iNOS inhibition was also observed when breast MDA-MB231 and prostate PC-3 cancer cells were subjected to ALA/light stress. We have now examined the signaling events associated with iNOS-mediated hyperresistance of COH-BR1 cells using a combination of immunocytochemistry, western blotting, immunoprecipitaion, and iNOS-kd methods. A soluble guanylyl cyclase inhibitor failed to stimulate ALA/light-provoked apoptosis, ruling out cGMP involvement in stress resistance. ALA/light activated PI3-kinase-dependent signaling via phosphorylation-inactivation of the tumor suppressor PTEN, leading to phosphorylation-activation of pro-survival Akt. Inhibition of PI3K by Wortmannin prevented ALA/light-induced Akt activation as well as iNOS upregulation while enhancing apoptotic photokilling. Moreover, photostress activation of PI3K/Akt was accompanied by a cytosol-to-nucleus translocation of the iNOS transcription factor NF-κB, and an inhibitor of NF-kB activation prevented iNOS induction while stimulating apoptosis. Furthermore, iNOS-kd as well as 1400W treatment resulted in intensified and more prolonged activation of MAP kinases JNK and p38α by ALA/light stress. In addition, photostressed iNOS-kd cells exhibited p53 upregulation and Survivin inactivation/downregulation. The following general mechanism of NO-based cytoprotection is suggested from these and related findings: Photostress activation of PI3K/Akt suppresses pro-apoptotic MAP kinases and p53 while upregulating/activating Survivin via NF-κB-mediated induction of iNOS/NO. This study provides important new insights into photostress-elicited pro-survival signaling that could reduce PDT effectiveness, and suggests iNOS inhibitor-based interventions for counteracting this.

(Supported by NIH Grant CA70823 and a grant from the MCW Cancer Center.)
Abstract A51: Antiproliferative activity of F14512, a novel polyamine-vectorized drug, on multidrug resistant cancer cells
Authors: Créancier Laurent , Guilbaud Nicolas , Annereau Jean-Philippe , Brel Viviane , Marionneau-Brossard Marie-Laure , Offrete Vanessa , Verdier Jérôme , André Karine , Fournier Emmanuel , Guminski Yves , Kruczynski Anna
Published: March 2013

F14512 is a novel vectorized topoisomerase II inhibitor currently in phase I clinical study in AML. Briefly, F14512 combines a polyamine (spermine) chain with an epipodophyllotoxin warhead. An enhanced antitumor effect associated with a large therapeutic index results from this specific targeting of cancer cells through polyamine transport system. One of the most important limitations in chemotherapeutic treatment is acquired or innate drug resistance. To investigate this phenomenon for F14152, we selected A549 NSCLC cell lines upon F14512 treatment by increasing doses selection, and characterized the profile of resistance in term of ABC transporter expression and modification of topoisomerase II expression. As results, resistant cell lines were selected after 46 passages and 32 treatment of F14512 with doses ranging 0.25 to 4 IC50 of inhibition of proliferation, over a period of 9 months. As key observation, the intense selection pressure led solely to a moderated shift of EC50 of proliferation, not mediated through MDR1 nor MRP1, but more likely attributable to reduction of topoisomerase II expression. Moreover, we demonstrated that F14512 retains significant antiproliferative activity of F14512 on cell line overexpressing MDR1: CEM selected in vinblastine, A-549 in vinflunine, and P388 in vinorelbine, with a striking enhanced impact on the later model. According to these first investigations, we conclude that MDR1 would not be involved in the resistance mechanism against F14512. We are investigating hypotheses potentially explaining the collateral sensitivity to this novel polyamine derivative on MDR1 expressing P-388 cells.
Abstract A53: Bayesian Network Inference Modeling Reveals Novel Regulators of Cell Cycle Progression and Survival
Authors: Gendelman Rina , Xing Heming , Sarde Preeti , Mirzoeva Olga K. , Feiler Heidi , Gray Joe W. , McDonagh Paul , Khalil Iya , Korn W. Michael
Published: March 2013

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate non-intuitive behaviors. We applied a novel computational strategy to decipher causal relationships between signaling network components. Gene networks were constructed from global gene expression profiles to model G1-S cell cycle arrest as a consequence of inhibition of MEK, a key component of the oncogenic RAF-MEK-ERK signal transduction pathway. Simulation of causally driven models generated a ranked list of 12 testable hypotheses, which confirmed role of known cell cycle regulators (CCND1, CCNE2, and CDC25A) and identified novel drivers of G1-S transition (IER2, TRIB1, C14ORF133). Validation confirmed 11 out 12 predictions. Second model which inferred causal relationships between genes lead to discovery of TRIB1 (tribbles homolog of Drosophila) as a regulator of NFkB-dependent gene expression. TRIB1 also mediates cross-talk to the NFκB pathway by enhancing p100 and IκBα-phosphorylation and degradation thus regulating cell survival and expression of cytokines crucial for tumor-stroma interaction. In agreement with this, knock-down of TRIB1 results in induction of apoptosis and sensitizes cells to cell killing induced by the death-receptor agonist TRAIL via upregulation of TRAIL receptor, DR5. Thus TRIB1 is a central regulator of cell cycle and survival and represents a potential therapeutic target. Therefore, applying REFS (Reverse Engineering Forward Simulation) to analyze gene expression profiles maximizes our ability to extract knowledge from experimental data.
Abstract A57: Chondroitin sulfate proteoglycan 4(CSPG4) Mediates Melanoma Cell Survival During PI3K and mTOR Inhibition
Authors: Javaid Sehrish , Wanshura Leah E. Colvin , Dudek Arkadiusz Z.
Published: March 2013

Background: Early development of resistance to therapy remains a major cause of cutaneous malignant melanoma treatment failure. Chondroitin sulfate proteoglycan 4 (CSPG4) is robustly expressed in a majority of metastatic melanomas. Previous work has shown CSPG4 to promote MAPK, PI3K and FAK activation, suggesting that CSPG4 represents a potential avenue for oncogenic growth and cell survival in response to chemotherapy. The PI3K-AKT-mTOR pathway regulates the cell growth and motility in the presence of nutrients. The inhibition of AKT and mTOR independently was shown to reduce the melanoma cell survival and enhanced the sensitivity of these cells to cisplatin. PF-05212384 (PKI-587) is a dual PI3K/mTOR inhibitor in clinical trials. We hereby report that the expression of CSPG4 on the melanoma cells caused them to become less responsive to the therapy with a dual PF-05212384.

Material and methods: Melanoma cell lines that stably expressed CSPG4 were selected for this study. The expression of CSPG4 was knocked down by using lentivirus-transduced shRNA. Cell survival assays were performed to compare the inhibitory effects of the drug in CSPG4 expressing and non-expressing cell line. Wound healing migration assays were performed to examine the cell motility and migration. The activation of PI3K-mTOR pathway was analyzed by phosphorylation of downstream effectors for their activation using Western blotting.

Results: Melanoma cells expressing active CSPG4 showed less sensitivity to the inhibitor. The average cell viability after treatment with maximum dose was 47.94% and 35.76% for CSPG4 positive 1205LU and WM1341D melanoma cell lines respectively, while the CSPG4 negative WM1552C showed an average cell viability of 19.07%. The difference in cell viability was statistically significant.(p<α=0.05)for higher doses. The suppression of CSPG4 using the shRNA sensitized the CSPG4 positive cell lines to the PI3K/mTOR inhibition. (p<α=0.05). The maximum effect was seen in the WM1341D where the cell viability was reduced at maximum dose to 26.30% as compared to 50% in non-targeting shRNA transduced control. The CSPG4 positive cell lines resisted the deactivation of p70Sk, a downstream effector of mTOR and had enhanced phosphorylation of transcription repressor 4E-BP1.At higher doses the cleavage of caspase to its activated forms, which is necessary for execution of apoptosis was reduced in the CSPG4 positive cell lines. The CSPG4 positive cells showed more motility as compared to the controls.

Conclusion: This data supports the hypothesis that the CSPG4 is involved in inducing resistance in melanoma cells in response to the therapeutic challenge with the dual PI3k/mTOR inhibitor. The study highlights the need of using multiple treatment strategies to treat melanoma.
Abstract B10: Overcoming resistance to B-Raf targeting agents using LPD nanotechnology
Authors: Chung Chin-Ying , Wang Yuhua , Sheng Wei-Yun , Tseng Yu-Cheng , Dick Taryn , Huang Leaf , Robertson Gavin
Published: March 2013

Constitutively active mutant V600EB-RAF is present in 50% to 60% of sporadic melanomas and vemurafenib is a selective inactivating inhibitory drug. While the compound is initially effective in 80% patients, most of the patients eventually developed more invasive resistant disease. Unraveling the mechanisms leading to the development of vemurafenib resistance is key to developing better treatment strategies that can overcome the process and provide patients with more effective treatment options. Here we show that vemurafenib inhibits ERK activity to negatively regulate tumor suppressor Sprouty4 expression to increase Akt3 activity, leading to the development of drug-resistant melanomas. Combining vemurafenib with sigma receptor targeted LPD nanoparticle technology containing Akt3 siRNA and suppressor Sprouty4 plasmid simultaneously inhibit MAPK and Akt3, preventing resistant tumor development in xenografted mice. Our studies have identified a novel mechanism leading to the development of resistant melanoma following vemurafenib treatment and development of a novel melanoma cell targeting nanotechnology-based therapeutic agent for combating the development of resistance.
Abstract B13: Modeling BRAF inhibitor resistance in genetically engineered mouse models of melanoma
Authors: Kwong Lawrence N. , Jiang Shan , Chin Lynda
Published: March 2013

Resistance to BRAF inhibitors in melanoma poses a serious clinical challenge to this otherwise potent therapy. Acquired resistance has been shown to arise from a wide variety of genetic and epigenetic sources including NRAS or MEK mutations, elevated PDGFRB or IGF1R levels, and BRAF splice variants. Many of these mechanisms were discovered in vitro and validated in patients, demonstrating the faithfulness of cell culture data. Nevertheless, cell culture may not be able to capture all sources of resistance, particularly if they involve interaction with the stroma. We have therefore undertaken the generation of de novo BRAF inhibitor resistance in a novel mouse model of melanoma, the iBIP (inducible Braf, Ink/Arf, Pten) model. Similar to existing BRAF mouse models, topical tamoxifen restricts BRAFV600E expression and PTEN loss to targeted melanocytes. The added utility of this model is the ability to extinguish BRAF transgene expression via withdrawal of doxycycline from the diet. We show that the molecular profile and tumor regression phentoype of BRAF inhibitor treatment in this model faithfully mirrors genetic BRAF extinction. Treatment with the BRAF inhibitor caused stable and complete tumor regression. However, long-term treatment with the inhibitor failed to generate de novo resistant melanomas. We hypothesized that the relatively stable mouse genome was not generating potentially resistance-conferring mutations or alterations at a fast enough rate. We therefore generated genomic instability in the iBIP model by crossing it onto a telomerase-deficient background. Melanomas in this model acquired resistance at a rate of approximately 30% after a short interval of sensitivity. Currently, characterization of these tumors is being carried out, including whole-exon sequencing, RNA-sequencing, and array comparative genomic hybridization. Preliminary aCGH data show a recurrent, whole-chromosome gain in two resistant tumors that is not present in untreated or sensitive tumors. Other efforts are focused on generating a large bank of resistant tumors, checking cross-resistance among BRAF inhibitors, and identifying any stromal and immune changes. This in vivo model of acquired BRAF inhibitor resistance provides an important complement to known resistance mechanisms and may also serve as a preclinical platform to test anti-resistance countertherapies.