bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B26: A requirement for p63 function in squamous cell carcinoma in vivo: Can the pathway be targeted for therapy?
Authors: Ramsey Matthew R. , Wilson Catherine , Faquin William C. , Mills Alea A. , Ellisen Leif W.
Published: June 2012

Squamous cell carcinoma (SCC) is a common form of human cancer that can arise in various epithelial tissues such as the lungs, skin, and head and neck region. Morbidity and mortality rates for SCC are high compared to many other cancers, and relatively little progress has been made in treatment due to a lack of molecular understanding of the disease. The p53-related protein p63 is essential for the formation of various epithelial tissues, such as the skin, and has many reported roles including suppression of apoptosis, maintenance of stem cell regenerative potential, regulation of differentiation, and maintenance of cellular adhesion. In SCC, p63 is overexpressed and has found to be the target of genomic amplification suggesting an important role in this disease. Previous in vitro studies have shown that p63 is required for survival of SCC cells in culture, however, cell lines lack the complex microenvironment found in human cancer, and thus the in vivo relevance of these results is unclear. To determine the role of p63 in vivo, we have generated a murine model of SCC, which faithfully recapitulates many of the molecular and pathological features of human SCC including invasion and metastasis. To test the role of p63 in tumor maintenance we crossed this model to a conditional p63-null allele (p63flox) and a tamoxifen-inducible Cre transgene, K14-CreER. We have found that genetic excision of both copies of p63 in established SCC tumors results in rapid and dramatic regression of tumors. In addition, even loss of a single copy of the p63 gene results in a reduced rate of growth, suggesting that SCC tumors are exquisitely sensitive to levels of p63 protein. These results support the idea that the p63 pathway is critical for the survival of SCC tumors in vivo, and targeting this pathway in SCC may result in more effective therapy for this highly refractory tumor type.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B26.
Abstract B28: Molecular basis of aberrant expression of alkaline phosphatase in renal brush border membrane from renal cell carcinoma patients
Authors: Sharma Ujjawal , Singh Sarwan Kumar , Prasad Rajendra
Published: June 2012

The incidence of renal cell carcinoma (RCC) has been increasing worldwide. It continues to present a diagnostic and therapeutic challenge. It accounts for approximately 3% of adult malignancies and 90% to 95% of neoplasm arising from kidney. RCC originates from proximal tubular epithelium of adult kidney. Alkaline phosphatase is abundantly expressed on the BBM and serves as an excellent marker for its integrity. Earlier reports have demonstrated that the activities of brush border membrane (BBM) enzymes are altered in cancer cells. The present study was conducted to find out the molecular basis of altered expression/activity of ALP in BBM from RCC in comparison to normal renal BBM. Total 30 histopathologically confirmed cases of RCC were included in the present study. The specific activity of ALP was drastically reduced in homogenate as well as in BBM from RCC as compared to BBM from adjacent normal kidney parenchyma. SDS-PAGE study showed that the BBM proteins of higher molecular weights were poorly expressed in BBM from RCC as compare to BBM from adjacent normal kidney parenchyma. Incubation of gel with BCIP/NBT dye showed that the expression of ALP in BBM from RCC was reduced as compared to adjacent normal kidney BBM. Western blot analysis using anti-ALP antibody also confirmed the reduced expression of ALP in RCC BBM. Further, to check whether this reduced expression of ALP occurs at mRNA level, a semiquantitative RT-PCR was done, which showed markedly reduced expression of ALP mRNA in RCC as compared to adjacent normal kidney.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B28.
Abstract B29: Genetic interactions between AKT/mTOR and Ras/MAPK pathways in liver cancer pathogenesis: from animal models to targeted therapy
Authors: Chen Xin , Ho Coral , Wang Chunmei , Lee Susie , Evert Matthias , Dombrowski Frank , Calvisi Diego
Published: June 2012

Introduction: Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) are deadly malignant liver tumors lacking of efficient treatment options. Akt/mTOR and Ras/MAPK pathways have been implicated in hepatic carcinogenesis. Our study is to define the genetic interactions between Akt/mTOR and Ras/MAPK pathways in liver carcinogenesis.

Methods: Activation of Akt/mTOR and Ras/MAPK signaling was assayed in 62 human HCC and 25 CCC samples. An activated Akt (myr-Akt) was stably transfected into the mouse liver alone or together with activated N-Ras (RasV12) via hydrodynamic gene delivery. Molecular and biochemical features of liver lesions were analyzed. A primary cell line (Akt/Ras) was isolated from a tumor co-injected with Akt and Ras protooncogenes. Akt/Ras cells and human HCC cell lines were treated with different mTOR inhibitors, including NVP-BEZ235, PP242 and Rapamcyin, as well as the MEK inhibitor AZD6244.

Results: Using human HCC samples, we demonstrated the coordinated activation of Akt/mTOR and Ras/MAPK pathways in subsets of HCC and CCC characterized by poor prognosis. We found that coexpression of myr-Akt with RasV12 synergizes to promote liver tumor development in mice, leading to HCC and CCC formation within 4 to 6 weeks post injection. At cellular level, tumors from Akt/N-Ras co-injection cells are characterized by high proliferation and low apoptotic index, and disrupted expression of genes involved in cell cycle regulation. At the molecular level, liver tumors show high levels of activated Akt/mTOR and Erk cascades. A primary cell line (Akt/Ras) was isolated from a tumor and used to test the response to different mTOR and MEK inhibitors. We found that dual PI3K/mTOR inhibitor NVP-BEZ235 has superior activity inhibiting Akt/Ras cell proliferation with IC50 around 2nM. It also efficiently inhibits phospho-S6, phospho-4EBP1 and cyclin D1 expression in Akt/Ras cells. Furthermore, NVP-BEZ235 synergizes with the MEK inhibitor AZD6244 to dramatically reduce the growth of the Akt/Ras cell line. Equivalent results were obtained in human HCC cell lines with the same inhibitors.

Conclusion: Coordinated activation of Akt/mTOR and Ras/MAPK signaling is the hallmark of aggressive subtypes of human HCC and CCC. Activated Akt cooperates with activated Ras/MAPK to rapidly promote liver tumor formation in mice. The Akt/Ras mouse model is a useful pre-clinical system to test anti-neoplastic therapeutic approaches against HCC and CCC in vivo. In addition, our preliminary studies suggest that the combination of a dual PI3K/mTOR inhibitor with a MEK inhibitor may represent a promising novel targeted therapy for treating human HCC harboring activated Akt/mTOR and Ras/MAPK pathways.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B29.
Abstract B30: Altered response to the prodrug of gemcitabine LY2334737 by overexpression or knockdown of carboxylesterase 2 (CES2)
Authors: Durland-Busbice Sara , Pratt Susan E. , Shepard Robert L. , Iversen Philip W. , Kays Lisa , Ballard Darryl , Heinz-Taheny Kathleen , Dantzig Anne H.
Published: June 2012

Gemcitabine (gem) is a nucleoside anticancer agent that inhibits cell cycle in S-phase and is registered clinically for the treatment of a number of solid tumors. A prodrug of gemcitabine, LY2334737, was developed for oral administration for metronomic dosing with the aim of growth inhibition of more tumor cells as they go through the cell cycle. LY is well absorbed and systemically cleaved in humans. The rate of hydrolysis is slow resulting in circulating levels of LY that are 10-fold higher than gemcitabine in plasma. Studies with cell lines and recombinant protein indicate that LY is cleaved to gemcitabine by carboxylesterase 2 (CES2). Because LY circulates in the plasma for an extended period of time, we wondered if expression of CES2 by the tumor could alter its response to the prodrug. The present study was undertaken to determine if the expression of CES2 alters cellular response to LY in vitro and in vivo. The constitutive expression of CES2 in SKOV-3 was knocked down by shRNA. A stable subclone was obtained that had CES2 expression reduced by 80%.

Stable HCT-116 transfectants were prepared that overexpressed CES2. Cell lines, transfectants and knock down cells were evaluated for drug sensitivity in a cytotoxicity assay. They were also evaluated for CES2 expression by biochemical cellular assay, Western analysis, and qRT-PCR. A xenograft study was conducted employing transfected HCT-116 cells. Immunohistochemical (IHC) staining was used to evaluate cell lines and tumors for CES2 expression. LY was less cytotoxic to SKOV-3 cells when CES2 was knocked down while sensitivity to gem was unaltered. Transfection of CES2 into low CES2 endogenously expressing HCT-116 cells enhanced LY drug sensitivity with no change in gemcitabine sensitivity. Stably transfected CES2 and mock HCT-116 transfectants were grown as a xenograft. The transfectants had identical growth rates in vivo and were equally sensitive to gem treatment. IHC staining of the tumors indicated that CES2 expression was maintained throughout the growth period. CES2 expressing tumors demonstrated a statistically significant greater response to LY than mock transfectant tumors. Taken together, these studies indicate that CES2 cleaves the prodrug and tumor expression of CES2 can enhance response to treatment with LY2334737.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B30.
Abstract B31: Potential for patient tailoring for prodrug of gemcitabine (LY2334737) by assessment carboxylesterase II expression in solid tumor biopsies
Authors: Holzer Timothy R. , Heinz-Taheny Kathleen , Ballard Darryl W. , Powell Eric L. , Dolled-Filhart Marisa P. , Christiansen Jason , Gustavson Mark , Biryukov Jennifer , Virayah Jagatheswari , Pratt Susan E. , Shepard Robert L. , Busbice Sara C. Durland- , Dantzig Anne H.
Published: June 2012

Carboxylesterase II (CES2), a serine ester hydrolase, is the major carboxylesterase responsible for the conversion of the prodrug of gemcitabine into its active form, gemcitabine (LY2334737), an anticancer chemotherapeutic. In humans, the ratio of plasma levels of prodrug to active form is 10:1. Therefore, increased availability of LY2334737 can result in cleavage of the prodrug into its active form at the tumor site and has potential for greater tumor cytotoxicity. Relatively high levels of expression of CES2 within non-neoplastic human tissue occur in the liver, kidney and gastrointestinal tract as visualized by immunohistochemistry (IHC). CES2 is also reported to be expressed in human cancers such as non-small cell lung carcinoma and colon adenocarcinomas. In this study, we analyzed CES2- transfected in vitro cell lines by real-time PCR, chromogenic IHC, and automated quantitative analysis (AQUA) of immunofluorescent in situ protein expression and showed that high levels of CES2 correlate with higher cytotoxicity. qRT-PCR analysis revealed that the mRNA levels of CES2 measured in formalin-fixed paraffin-embedded tumors correlate with protein levels. CES2 transcriptional profiling was performed to identify additional tumor types with high levels of expression of CES2. CES2 overexpression was detectable by IHC and/or AQUA analysis in human colon carcinoma, mesothelioma, non-small cell lung cancer, and tumors of the breast and ovary. CES2 IHC labeling in colonic adenocarcinomas was diffuse. However, labeling in non-neoplastic mucosa showed low levels in proliferating cells at the crypt base and increasingly higher expression toward the terminally differentiated cells at the tips—cells that would not progress through the cell cycle and should not be affected by antiproliferative agents. In conclusion, we have developed robust IHC and AQUA-based assays for differential expression of CES protein levels in a variety of archival human tumor types and have shown high correlation with CES2 transcript levels by qRT-PCR. These methodologies can identify tumors with high levels of CES2, a biomarker that may further be investigated in biomarker-driven clinical trials to identify patients who will more likely respond to LY2334737.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B31.
Abstract B32: TGF-β blockade improves the distribution and efficacy of chemotherapeutic agent in breast carcinoma
Authors: Liu Jieqiong , Liao Shan , Huang Yuhui , Jain Rakesh K. , Xu Lei
Published: June 2012

In its journey from blood vessels to cancer cells, a therapeutic agent must overcome the transport barrier posed by the interstitial matrix. In contrast to our understanding of blood vessels, our understanding of the formation and function of the tumor interstitial matrix is still in its infancy. A primary component of the interstitial matrix, collagen type I, has been shown to inversely correlate with diffusive transport in solid tumors. TGF-β can stimulate production of collagen by stromal cells in tumors. Thus, we studied the effect of blocking TGF-β pathway in breast carcinoma progression and response to chemotherapy. To this end, we used a soluble Fc:TGF-β type II receptor fusion protein (sTβRII) to block TGF-β pathway in two breast cancer models (human MDA-MB-231 cells and mouse 4T1 cells). TGF-β blockade significantly impaired the growth and metastasis of orthotopic tumors in both tumor models. The recruitment of myofibroblasts and interstitial matrix content was determined by immunohistochemistry, TGF-β blockade significantly decreased the recruitment of myofibroblast and the level of collagen I. Next, to determine the effect of TGF-β blockade on interstitial transport, we injected fluorescent doxorubicin into the tail vein of mice and observed the intratumoral distribution of doxorubicin autofluorescence by confocal microscopy. We found that TGF-β blockade increased the transvascular flux of fluorescent doxorubicin. As a result, doxorubicin liposome (DOXIL) treatment was significantly more effective in inhibiting tumor progression in TGF-β blocked tumors compared to parental tumors. In summary, our study characterized the functional role of TGF-β in modulating tumor interstitial matrix in breast cancer xenografts and suggested that blocking TGF-β signaling pathway may represent a testable therapeutic approach to be combined with conventional chemotherapy in breast cancer treatment.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B32.
Abstract B33: Intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a novel prodrug activator gene transfer technology in an immune-competent model of glioblastoma
Authors: Ostertag Derek , Amundson Karin , Buckley Taylor , Lopez Fernando E. , Martin Bryan , Gruber Harry , Gunzburg Walter , Perez Omar , Jolly Douglas J. , Robbins Joan M.
Published: June 2012

Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have poor prognosis and current treatments remain only palliative. Previous gene therapy approaches using replication-deficient viral vectors have demonstrated limited efficacy likely due to poor gene delivery to the tumor.

Methods: We used a novel replication-competent retroviral vector (Toca 511) designed and engineered to efficiently deliver a modified cytosine deaminase (CD) prodrug activating gene to glioma cells. Previous studies showed that such a replicating vector can preferentially infect intracranial human xenografts in nude mice (Tai et al. Mol Ther. 2005;12:842) and lead to increased survival in treatment groups compared to controls. In preparation for clinical trials we have improved the CD gene activity and further stabilized the vector structure to create Toca 511. We investigated the functionality of Toca 511 using stereotactic, intratumoral delivery in a syngeneic, orthotopic glioma model in immune-competent B6C3F1 mice. Freshly excised tumors were analyzed by quantitative PCR, Western, and HPLC.

Results: Brain tumors isolated from mice treated with Toca 511 and fluorocytosine (5FC) had observable expression of CD protein and vector copy numbers per genome that demonstrate efficient tumor infection. Extensive tumor specific viral spread with almost no spread of vector from the site of injection was observed in the first 24 days. Highly efficient intratumoral conversion of the 5FC prodrug into the anticancer drug 5-fluorouracil (5FU) was demonstrated after intraperitoneal or oral dosing with 5FC. Studies with Toca 511 at high and mid dose levels in combination with 5FC resulted in prolonged survival compared to controls (> 5 months, p<0.0036 and <0.0354 respectively) in this syngeneic glioma model. The highest Toca 511 dose level tested in combination with 5-FC yielded no safety related findings in the mice.

Conclusions: Toca 511 efficiently delivers a functional CD gene for expression in glioma cells in a syngeneic, orthotopic model in immune-competent mice. Toca 511 increases survival through the delivery of CD gene and subsequent efficient conversion of 5FC into 5FU within the brain tumor with no safety related findings. A phase 1 ascending dose trial investigating the safety and tolerability of Toca 511 in combination with 5FC in patients with recurrent GBM is about to initiate.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B33.
Abstract B34: Inhibition of lung cancer cell invasion by siRNAs delivered by cell-specific aptamers
Authors: Wang Wei-Ya , Lai Wei-Yun , Hsiao Tzu-Hung , Peck Konan
Published: June 2012

Cell-specific delivery of therapeutic agents and targeted therapy are highly pursued topics in medical researches for treating cancers and other diseases. We aim to develop technologies enabling delivery of siRNA to cancer cells to retard or inhibit metastasis of the cancer cells with minimal adverse effects to normal cells. As long as the cancer cells do not invade and are confined, they can be eradicated by means such as surgical resection or radiation therapy.

Oligonucleotides of different sequences can fold into different conformations to bind to their targets with high specificity and affinity. These structured oligonucleotides are known as aptamers. We have identified new surface markers which prevalently expressed on lung cancer cells. By identifying functional aptamers against the surface marker proteins, we show that once the marker protein is activated by the aptamer, the surface marker protein/aptamer complex internalizes into the lung cancer cells. Therefore, it is feasible to employ the functional aptamer as a carrier to deliver siRNA into cancer cells to silence the expression of genes associated with metastasis. The aptamer-siRNA chimera is formed by chemical synthesis and bioconjguation methods. The chimera consists of an aptamer, which delivers therapeutic siRNA specifically to cancer cells but not normal cells to knock down the target genes that are pivotal in signaling pathways associated with the events of metastasis cascade. The effective knockdown of the metastasis associated genes by the nucleic acid chimera is verified by employing a series of invasion assays such as wound healing, Matrigel, filopodia formation, and others. The in vitro experimental results show that the aptamer-siRNA chimera is able to deliver therapeutics to specific target cells and silence the expression of the target genes in the cells. The in vitro experimental results are further verified by in vivo xenograft mouse model with immunohistochemistry examination, tumor size measurement, and circulating tumor cell detection by use of Alu sequence as the marker. By using lung cancer as a model, the data indicate that it is durable to employ the aptamer-siRNA chimera for cell- and target-specific therapy of cancer metastasis.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B34.
Abstract B36: Identification of carboxylesterase 2 as a hydrolase that cleaves the prodrug of gemcitabine LY2334737
Authors: Pratt Susan E. , Durland-Busbice Sara , Shepard Robert L. , Harlow Harry B. , Ballard Darryl , Heinz-Taheny Kathleen M. , Dantzig Anne H.
Published: June 2012

Gemcitabine hydrochloride is a nucleoside anticancer agent registered for the treatment of pancreatic, NSCLC, breast, and ovarian cancers. A prodrug of gemcitabine (LY2334737) was prepared that is a gemcitabine analog with an amide-linked valproate. This prodrug is noncytotoxic and must be hydrolyzed to release gemcitabine. In man, the prodrug is orally absorbed intact and converted to gemcitabine systemically. This study was undertaken to identify the enzyme responsible for hydrolysis of the prodrug to gemcitabine. The NCI-60 cell line panel was screened for prodrug sensitivity and microarray data were used to evaluate the expression of hydrolase enzymes in responsive and unresponsive cell lines. Cell extracts and candidate enzymes were tested for LY hydrolysis using HPLC and cell lines were evaluated for enzyme expression using qRT-PCR and Western methods. Screening of the NCI-60 panel demonstrated that LY was not cytotoxic to the majority of cell lines, however sensitivity was observed in a few cell lines such as SK-OV-3 and COLO205. Other cell lines were also evaluated. Extracts of drug sensitive cell lines hydrolyzed LY to gemcitabine and its inactive metabolite 2',2'-difluorodeoxyuridine (dFdU), indicating that LY hydrolysis was necessary for activity. Analysis of the expression of known hydrolases in the NCI-60 microarray data identified the serine ester hydrolase, carboxylesterase 1 (CES1) among the top candidates. In humans, there are 3 CESs and these are known to hydrolyze other prodrugs. Human recombinant protein of each CES was tested for LY cleavage. With long incubation periods, only CES2 hydrolyzed the drug. Loperamide is an inhibitor of CES1 and CES2 that is permeable to cells and noncytotoxic. At low micromolar concentrations this inhibitor only affects CES2, therefore cytotoxicity assays were conducted with SK-OV-3 in the presence and absence of 10 µM loperamide. The cytotoxicity of LY was decreased with loperamide CES2 inhibition without effect on gemcitabine cytotoxicity. Expression of CES2 was evaluated in drug-sensitive and -insensitive cell lines by qRT-PCR and Western analysis; LY sensitive lines expressed CES2 while insensitive lines did not. An immunohistochemical assay was developed that showed expression of CES2 to be heterogeneously expressed in these cell lines. Taken together, these data indicate that CES2 hydrolyzes the prodrug. Cancer cells that express CES2 may have an enhanced response to treatment with the prodrug LY2334737.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B36.
Abstract B37: Prognostic analysis of primary hepatic carcinoma with cancer embolus of biliary duct
Authors: Zhou Bao Tian
Published: June 2012

Background: In China, primary hepatic carcinoma (PHC) is one of the most frequent malignancies, which is more accompanied by cancer embolus of portal vein and is less with cancer embolus of biliary duct (CEBD) clinically. Anterior cases were diagnosed as PHC with CEBD by B ultrasound, CT, MRCP, or ERCP. Hepatectomy and choledochotomy were performed resecting the primary tumor and cleaning the CEBD completely, followed with TACE after operation. Nowaday ultrasound in biliary duct and orthotopic liver transplantation were worth considering as a brand new and extreme diagnose and treat method respectively. The article was intended to evaluate the clinicopathological characteristics and the prognosis of PHC with CEBD by therapeutic effect contrast to PHC with CEBD in different location.

Methods: The clinical data of 412 PHC patients with CEBD underwent surgical hepatectomy from 2000 to 2009 in the several hepatobiliary surgery centers of hospitals in China were retrospectively analyzed to research the clinicopathological characteristics and the prognosis. All the patients were divided into three groups based on distribution and location of CEBD.

Results: The overall 1 -year, 3-year, and 5-year survival rates from the time of operation were 76.8%, 53.4%, and 40.1 %, respectively. There was no significant difference in the prognosis between PHC patients with or without CEBD (P>0.05). Independent factors affecting the prognosis of PHC patients were infiltration growth and multiple development. CEBD had no contribution in the prognosis of PHC patients. But the prognosis was different in PHC patients with different locations of cancer embolus in the biliary duct (P<0.05).

Conclusion: Surgical operation remains the most effective treatment to PHC patients with CEBD. CEBD had no contribution in the prognosis of PHC patients. The location of the cancer embolus in the biliary duct was significant factors affecting the prognosis of PHC patients with CEBD. The prognosis was the worst in those PHC patients with CEBD located in the right hepatic duct.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B37.
Abstract B4: Chromosome 17p13.1 (KCTD11), a smoking-related susceptible region linked to lung cancer risk in Korean populations
Authors: Lee Yeiwon , Lee Minhyeok , Cho Hyunjung , Hong Jeeyoung , Kwon Seonjung , Park Chang-Gyo , Lee Hoi-Young , Son Ji-Woong , Kang Jaeku , Hong Jeeyoung
Published: June 2012

Lung cancer is the most common cause of cancer death worldwide. Smoking is known as the strongest single factor for the development of lung cancer. However, there are inherited genetic factors that cause different responses to cigarette smoking exposure among individuals. We tried to identify these differences in heavy smokers by examining copy number variations (CNVs) between lung cancer patients and healthy controls. Analysis by array comparative genomic hybridization (aCGH) showed 26 significant (P<0.05) clones with either copy number gains or losses. 3 genes, KCTD11, FGF11, and PTPRH on chromosomal regions 17p13.1 (KCTD11 and FGF11) and 19q13.42 (PTPRH) were selected (P<0.001) and tested by realtime quantitative PCR (qPCR) with 34 healthy controls and 54 lung cancer patients for large-scale analysis. KCTD11, a known tumor suppressor gene, showed copy number losses in cancer patients. In addition, as the years of smoking increased, odds ratios were also increased (0R=0.8,2.2, and 16.0), implying that it is a susceptible gene to lung cancer when an individual smokes. Therefore, KCTD11 is a promising candidate for a predictive marker to identify individuals with high, inherited genetic risk for the development of lung cancer.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B4.
Abstract B40: PALB2-KEAP1 interaction links oxidative stress to cancer and Fanconi anemia
Authors: Ma Jianglin , Xia Bing
Published: June 2012

PALB2/FANCN is mutated or silenced in breast, ovarian and pancreatic cancers and Fanconi anemia. It was initially discovered as a major binding protein for BRCA2 and controls the function of BRCA2 in homologous recombination, DNA double strand break repair and tumor suppression (Xia et al 2006). Nowadays, PALB2 itself has been also identified as a BRCA2-like breast cancer susceptibility protein and links BRCA1 and BRCA2 in the DNA damage response (Zhang and Ma et al 2009, Sy et al 2009). Therefore, PALB2 has been thought as an integral component of BRCA complex required for maintaining genomic stability via DNA recombinational repair.

In order to explore what other functions PALB2 protein may have, we performed a comprehensive search of PALB2-binding partners using the tandem affinity purification (TAP) assay combined with tandem mass spectrometry (LC-MS/MS) analysis. We show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the anti-oxidant transcription factor NRF2. NRF2-KEAP1 pathway has been characterized as an important endogenous mechanism for combating oxidative stress. Oxidative stress-induced damage can lead to a variety of pathologies such as cancer, Parkinson's diseases, diabetes, Alzheimer's diseases and the aging process (Valko et al 2007). We demonstrate that PALB2 shares a conserved “ETGE” motif with NRF2 and competes with NRF2 for KEAP1 binding, thereby relieving NRF2 from KEAP1-mediated repression. As such, PALB2 promotes NRF2 nuclear accumulation, protects cells against oxidative stress and reduces cellular level of reactive oxygen species (ROS). Our findings establish a new function for PALB2 and provide a direct link between oxidative stress and the predisposition of cancer and Fanconi anemia.

References:

1. Xia B et al. Mol Cell. 2006;22:719.

2. Zhang F et al. Curr Biol. 2009;19:524.

3. Sy S et al. Proc Natl Acad Sci USA. 2009;106:7155.

4. Valko M et al. Int J Biochem Cell Biol. 2006;39:44.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B40.
Abstract B41: Restoring sensitivity to anti-EGFR-based therapy in lung adenocarcinoma by downstream modulation of the FOXO1/KLF6 transcriptional network
Authors: Sangodkar Jaya , Katz Suzanna , Albano Tara , Melville Heather , Burstein David , Politi Katerina , Difeo Analisa , Narla Goutham
Published: June 2012

Anti-EGFR-based strategies have shown clinical efficacy in the treatment of metastatic lung adenocarcinoma. Nevertheless, treatment resistance invariably develops through either de novo activation of downstream RAS/AKT signaling or acquired mutations in the Epidermal growth factor receptor (EGFR). A more thorough molecular characterization of the downstream mediators of EGFR signaling may lead to the development of newer classes of targeted molecular therapies to treat resistant disease. Here we identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that regulate response to anti-EGFR-based therapies in both cell culture and in vivo models of the disease. Specifically, inhibition of AKT signaling promotes FOXO1 stabilization resulting in transactivation of the KLF6 tumor suppressor gene and induction of apoptosis in lung adenocarcinoma cell lines. Furthermore, the use of FDA-approved drug trifluoperazine hydrochloride, a FOXO1 nuclear export inhibitor restores sensitivity to erlotinib-resistant cell lines through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft lung adenocarcinoma models. In addition, quantitative real-time PCR-based analysis and Western blotting confirm a significant correlation between activated oncognic EGFR signaling and downregulation of the FOXO1 and KLF6 tumor suppressor gene network in both primary human lung adenocarcinoma patient samples and a transgenic mouse model of the disease. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify targeted molecular agents to restore chemosensitivity to anti-EGFR-based therapy in culture and in vivo.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B41.
Abstract B42: Molecular and cytogenetic characterization of novel multidrug-resistant human tumor cell lines
Authors: Ruzdijic Sabera , Podolski-Renic Ana , Pesic Milica , Chiourea Maria , Bankovic Jasna , Andjelkovic Tijana , Milinkovic Vedrana , Tanic Nikola , Gagos Sarantis
Published: June 2012

Multidrug resistance (MDR) is the major cause of failure to a successful cancer treatment. Cancer cells manifesting MDR phenotype are cross-resistant to many structurally and functionally unrelated drugs. The purpose of this study was to establish in vitro models, i.e., resistant cell lines, to further investigate the mechanisms of MDR. We used the human colon cancer cell line DLD1 and the glioma cell line U87 to establish paclitaxel-resistant cell lines, DLD1 -TxR and U87-TxR, by continuous exposure to stepwise increasing concentrations of the drug. The newly established, DLD1 -TxR and U87-TxR cell lines showed extensive resistance to paclitaxel (301-fold and 103-fold respectively). They also demonstrated cross-resistance to other anticancer agents such as doxorubicin, epirubicin, vinblastin, and etoposide. Cytogenetic data based on inverted-DAPI Banding, demonstrated that both resistant cell lines retained several karyotypic characteristics of their sensitive counterparts and also acquired novel structural or numerical chromosomal aberrations that may be related to resistance to chemotherapy. Interestingly, while the dividing parental U87 cells were mainly near-tetraploid, chemoresistance in the U87-TxR cell line was accompanied by one level ploidy reduction as verified by the increased prevalence of near-diploid cell populations. In addition, the DLD1 -TxR cells showed an increase tendency to loose the Y chromosome. Gene expression analyses revealed a 4-fold increase in the level of mdr1 mRNA in DLD1 -TxR cells. U87-TxR cells demonstrated prominent mdr1 activity since the expression in parental cells was undetectable. In contrast, the level of mrp1 mRNA was 40% decreased in both resistant cell lines. The expression of other MDR-related genes, topo IIα, and lrp, was unaffected by paclitaxel resistance. Flow cytometry analyses showed that the accumulation of rhodamine in resistant cells DLD1 -TxR and U87-TxR was significantly lower than in their sensitive counterparts, 17-fold and 8-fold, respectively. These results indicate that P-gp, encoded by mdr1 gene, is significantly overexpressed in both resistant cell lines and may account for paclitaxel resistance. In conclusion, the two novel resistant cell lines, DLD1 -TxR and U87-TxR may serve as appropriate models for the study of the mechanisms of paclitaxel resistance offering novel insights into potential targets for overcoming MDR that would improve the response to chemotherapy and patient outcome.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B42.
Abstract B43: Comparative proteomic analysis of colorectal cell lines to identify biomarkers for the prediction of intrinsic resistance to FOLFOX chemotherapy
Authors: Unger Florian T. , Meyer Markus , Spörl Monika , Harasym Agnieszka , Sullivan Kevin , Juhl Hartmut , David Kerstin A.
Published: June 2012

As there is a great interpatient variability in drug response caused by the individual geno- and phenotype the choice of optimal chemotherapy should be tailored for each patient. Intrinsic drug resistance is one of the main reasons for therapeutic ineffectiveness of first line therapy and recurrence in colorectal cancer. To advance individualized chemotherapy and understanding of drug resistance mechanisms, predictive biomarkers which indicate individual drug response of patients are urgently needed. The prediction of response to first line therapy will identify subgroups of patients who benefit from treatment and thus avoid ineffective treatment and associated side effects.

In this study, several commercially available cell lines as well as primary epithelial mixed cultures and clonal cell lines established from colorectal cancer patients were treated with different drugs, such as 5-fluorouracil, oxaliplatin, and drug combinations, e.g., FOLFOX. The determination of chemosensitivity of these cell lines to drug treatment was performed using two different assays, the ATPlite™-(Perkin Elmer) and FMC-assay (according to Larsson et al.). Both assays showed time- and dose-dependent drug responses which differed significantly among the 18 colorectal cell cultures. The analysis of obtained in vitro chemosensitivity data allowed us to cluster sensitive versus resistant cell lines by determining IC50-values. For the discovery of differentially expressed proteins which might be linked to a specific phenotypes of intrinsic chemoresistance we generated protein expression patterns from all 18 cell lines using a combination of reversed phase highperformance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (RP-HPLC MALDI MS). Up to 3500 m/z ion signals in the molecular mass range between 2500-35000 m/z were detected reflecting a complex pattern of small proteins and peptides. For protein identification, RP-HPLC fractionated samples were purified and digested by trypsin. Fragments of interest were subjected to MS/MS analysis in the TOF/TOF mode. The corresponding MS/MS spectra were used to search the NCBInr database using MASCOT software. Based on these data, we hope to gain more understanding of the biological processes of chemoresistance concerning important cellular drug responses (e.g., signal transduction pathways) and thereby obtain insight into new drug targets for future therapeutic intervention.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B43.
Abstract B44: Probing the effects of linker flexibility in second mitochondria-derived activator of caspases mimetic dimers and assessing their ability to induce apoptosis in a MDA-MB-231 cell line
Authors: Cheema Taz , Ben Robert , Cheung Herman , LaCasse Eric
Published: June 2012

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (lAPs). X-linked inhibitor of apoptosis protein (XIAP) is one of the best characterized lAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), in particular caspase −3, −7, and −9 through one of its baculovirus IAP repeat (BIR) domains. Second mitochondria-derived activator of caspases (SMAC) is an antagonist protein released from the mitochondria and into the cytosol, to disrupt the complexes produced between XIAP and caspase. SMAC binds to the BIR domains in XIAP through its tetra-peptide motif, AVPI. Studies have demonstrated that the first and third amino acid positions of this tetra-peptide motif are important for binding, while positions two and four can be modified to increase binding affinity (AXPX).

It has also been shown that dimers of SMAC mimetics show greater potency than their monomer counter parts. Previously, our laboratory has synthesized and tested SMAC dimers using three glycine amino acids to link AXPX sequences. These compounds showed very little activity. We also synthesized and tested SMAC dimers comprised of a lysine amino acid, linking two AXPX sequences; these mimetics did display significant activity. In particular an (AKPF)2K sequence showed 50% cell death at 10 uM when combined with death ligand TRAIL. Synthesis of these compounds was conducted using solid phase peptide synthesis (SPPS) on a polystyrene resin.

We hypothesized that flexibility in the linker joining the two AXPX sequences was necessary to increase binding affinity. Consequently several SMAC mimetic dimers built on two different linkers (gamma-amino butyric acid and ornithine), both of which have a four carbon spacer between each terminus, have been prepared and evaluated for activity using a MDA-MB-231 breast cancer cell line. The first series of SMAC mimetic compounds consists of MeAXPX-GABA-MeAXPX amino acid sequence while the second series of SMAC mimetics are comprised of a MeAXPX-Ornithine-XPXMeA amino acid sequence. All data and results, including synthesis, biological assays, and the testing of these compounds will be the major focus of this presentation.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B44.
Abstract B45: The short-term effects of arsenic on Cdk5-dependent apoptosis of prostate cancer cells
Authors: Chen Mei-Chih , Wang Paulus S. , Lin Ho
Published: June 2012

Arsenic compounds have been used widely in medicine to treat a variety of diseases. However, it has also been believed to involve the carcinogenesis of prostate cancer and the hormone disruption might be one of the causes. Our previous results indicate that digoxin treatment leads to apoptosis of prostate cancer cells through Cdk5 overactivation, in which the regular activator of Cdk5, p35, is cleaved into more potent one, p25. Here, our latest data showed that arsenic also caused p35 cleavage and subsequently Cdk5 overactivation in prostate cancer cells. Similarly, this arsenic- induced stimulatory effect on Cdk5 resulted in apoptosis of prostate cancer cells accompanying with the degradation of androgen receptor in LNCaP cells. This result implies that androgen is no longer a trophic factor for prostate cancer cells after arsenic treatment. Although STAT3 was identified by us as a Cdk5 substrate responding to tumor growth, arsenic-triggered Cdk5 overactivation did not increase STAT3 phosphorylation. In conclusion, although long-term exposure of arsenic possibly causes prostate cancer, our present results provide evidence indicating that in vitro and short-term treatments of arsenic might lead to apoptosis of prostate cancer cell lines through Cdk5 overactivation pathway. This study may arouse the new indication for the old chemical and also shed light on the application on cancer therapy.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B45.
Abstract B46: Proliferative suppression of anthraquinone derives emodin and aloe-emodin through selective targeting in prostate and breast cancer cells
Authors: Huang Pao-Hsuan , Lin Ho
Published: June 2012

Emodin and aloe-emodin are the main bioactive components of anthraquinones found in Chinese herbal medicine, Rhubarb (Rheum palmatum), and aloe-emodin is also abundant in the leaves of aloe vera. Although emodin and aloe-emodin are capable of inhibiting cancer cell proliferation or inducing apoptosis, the recent studies suggest that the molecular targets of these two anthraquinones are different. Emodin has been reported to suppress HER-2-overexpressing breast cancer cells proliferation through decreasing HER-2 tyrosine kinase activity. Besides, emodin affected androgen receptor (AR) transcriptional activity by preventing AR nuclear translocation and promoted AR degradation. Aloe-emodin has been found the antiproliferative effects on breast cancer cells which is through estrogen receptor (ER)-modulating ability. Interestingly, emodin and aloe-emodin share similar structure with different functions by hitting different targets. Therefore, the molecular mechanisms of aloe-emodin causing cancer cell death are interesting to explore. Here, three cancer cell lines were used in our experiments: LNCaP, a human prostate cancer cell line with AR expression; two human breast cancer cell lines: MDA-MB-453 with high expressions of HER-2 and AR; MCF-7 with low expressions of HER-2 and AR. In which, MCF-7 cells contain high expression level of ERα. Cells were treated with emodin or aloe-emodin for different time intervals and proliferation was determined. The results indicated that emodin decreased the proliferation of LNCaP and MDA-MB-453 which express high AR protein; however, MCF-7 with low AR protein were relatively insensitive to emodin treatment. Oppositely, aloe-emodin decreased cell proliferation in ER-positive MCF-7 cells but had no effects on LNCaP and MDA-MB-453 cells. Furthermore, the morphology of MCF-7 was dramatically changed after aloe-emodin treatment and cell death was apparently observed. The protein levels of HER-2, phospho-HER-2, and ErbB3 were decreased after aloe- emodin treatment while ERK was unaffected. In addition, the protein levels of ERα were reduced by aloe-emodin in dose-dependent manner; however, AR protein seems unaffected. In conclusion, our findings indicate that aloe-emodin selectively attacks targets in cancer cells and had potentials to treat HER-2/ER-positive breast cancer in the future.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B46.
Abstract B48: Immunohistochemistry and RT-PCR evaluation of fixative effects in a model tissue system
Authors: Millward Carl , Liu Mei-Lan , Jeong Jennie , Ambannavar Ranjana , Kaplan Hargita , Collin Francois , Baker Joffre , Cronin Maureen
Published: June 2012

The standard practice in hospital pathology laboratories is to preserve patient clinical tissue specimens as fixed, paraffin-embedded (FPE) tissue. FPE specimens are used for routine pathologic examination, immunohistochemistry (IHC) studies, and a variety of molecular diagnostic assays. The results of these studies assist in determining the patient's clinical status and in therapeutic decision making. However, the methodology for tissue fixation is not standardized across laboratories and a number of different tissue fixatives are currently commercially available. The use of different tissue fixatives may significantly affect the performance of IHC and molecular diagnostic assays.

The results of nine common tissue fixatives and their effects on both IHC- and RNA-based molecular assays are reported. Using human placenta as a model tissue system, nine common fixatives (B5, Bouin's, ethyl alcohol 70%, formalin, Hollandes, Penfix, Prefer, Zenker's, and zinc formalin) were compared for effects on six IHC assays and a panel of 42 gene targets by RT-PCR assays, as well as performance relative to fresh (RNAlater®) or frozen (OCT) unfixed tissue. The 42-gene panel assessed by RT-PCR included the six genes assessed by IHC. The IHC assays were scored using a semiquantitative method. For RT-PCR, raw assay scores were derived and subsequently normalized. Different fixatives resulted in varying effects on IHC and molecular assay performance. Per gene across each fixative, mRNA expression levels assessed by RT-PCR assays demonstrated wide variation, which could be largely corrected for by normalization. Variation in immunoreactivity as a function of tissue fixative was also observed with IHC assays. Compared to IHC, RT-PCR assays demonstrated greater sensitivity and were able to detect lower levels of gene expression, when the IHC assay gave negative results. Interestingly, fixative related effects were not always similar between IHC and RT-PCR assays. Therefore, it is recommended that the effects of tissue fixation be taken into consideration when performing data analysis and making comparisons between IHC and molecular diagnostic assays.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):B48.