bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of c-FLIP(long), c-FLIP(short), and caspase 8 expression in primary tumors using a semiquantitative multiplex LSC-based assay
Authors: Wall Jason A. , Thor Christopher , Cottrell Susan , Rand Hugh , Branstetter Daniel , Pan Yang
Published: May 2015

Recent studies have demonstrated the value in quantification of tumor expression and mutational status of growth factor signaling components as potential predictors of clinical response. Quantification of elements in the apoptosis pathways may provide a similar value in predicting clinical response to new therapies based on induction of apoptosis through death-receptor-mediated signaling. To that end, we have qualified a 5-color multiplexed immune histofluorescent tissue-based assay to provide semiquantitative measurement of expression of death-inducing signaling complex (DISC) components. The DISC components we measured in FFPE archival samples from human solid tumors are caspase 8, c-FLIP(long) and c-FLIP(short). Additional components of the multiplex were a pan-keratin reagent for identification of cytokeratin expressing epithelial tumor cells, and a histone H3 reagent for staining quality control. Image collection was accomplished via laser scanning cytometry (LSC) with a data pipeline which generates FCS3.0 (flow cytometry standard 3.0) files. This allowed intensity analysis of gated tumor enriched regions via standard flow cytometry analysis software. Antibodies were identified via in vitro screens using high/low target expressing cell lines, and further tested using xenografts of the cell lines of interest. The assay was qualified by examining day-to-day, slide-to-slide, and inter-operator variability using xenograft TMA (tissue micro-array) controls. The intensity of stained samples over 24 hours was stable. Total experimental %CV was demonstrated to be less than 25%. We analyzed 6 commercially available cancer TMA slides containing 387 tumor cores representing colon, lung, pancreas, breast, sarcoma, and non-Hodgkin's lymphoma, along with 43 corresponding normal cores. We found a wide range of expression of caspase 8, c-FLIP(l), and c-FLIP(s) within each tumor type, as well as clear differences between normal and tumor tissues. These findings indicate a potential for stratification of clinical response by DISC protein expression in therapeutics that act via death receptor mediated apoptosis.
Identification of complex oncogene signatures in human cancer specimens and cell lines
Authors: Ethier Stephen P. , Atikkuke Govindaraja , Haddad Ramsi
Published: May 2015

Activated oncogenes are the genetic drivers of human cancer progression. Oncogenes become activated by structural DNA alterations including gene amplification accompanied by gene over expression, point mutations, and gene translocations/rearrangements. Data from recent clinical trials using targeted agents strongly suggest that clinical efficacy of these drugs is optimal when the target is an activated oncogene. However, it is also clear that even among patients that respond to drugs that target activated oncogenes, the level and duration of response is highly variable. One possible reason for heterogeneity in response to oncogene-targeted agents is the genomic context of a particular activated oncogene is not the same in all patients. Thus, the genomic context of an oncogene-target can have a dramatic influence on drug efficacy. Therefore, the ability to detect and diagnose complex oncogene signatures in human cancer specimens is vital for better predictions of the efficacy of targeted agents, and to design personalized therapeutic strategies. In our Oncogenomics laboratory, we have been working to identify candidate amplified and point mutated oncogenes in human breast, ovarian and lung cancer specimens and cell lines. To obtain individual oncogene signatures, we have used high resolution array comparative genomic hybridization coupled with a novel gene expression algorithm that allows for the identification of genes that are over expressed within amplicons in individual specimens, and Sequenom analysis for identification of point mutated oncogenes. These approaches have allowed us to elucidate complex oncogene signatures for over 50 cancer cell lines and specimens. Analysis of these data sets suggests that oncogene combinations across specimens is nearly random. Thus, knowing the status of one oncogene such as HER-2 does not predict what other oncogenes may be activated in the specimen or cell line. Indeed, we have observed HER-2 amplifications in the context of amplifications of oncogenes in the 11q12 or 8p11 amplicons, among others, and in the presence of absence of PIK3CA mutations. Furthermore, analysis of the effects of small molecule inhibitors of HER-2 at the level of cell survival/proliferation and gene expression demonstrated a dramatic effect of oncogene context on response to a targeted agent. In particular, PIK3CA mutations in the presence of HER-2 amplification and over expression was associated with a greatly attenuated response of the cells to the HER-2 targeted agent. Collectively, these results point to a need for a molecular diagnostic platform that can accurately identify the causal driving oncogenes in an individual cancer biopsy specimen in order to optimize clinical trial design, and develop personalized therapeutic strategies.
Comparison of signal transduction drug response markers in immortalized cell lines and primary patient specimens
Authors: Ceceris Kyra D. , Ervin Paul R. , Cassino Theresa R.
Published: May 2015

Background: The epidermal growth factor pathway is an important driver of cancer growth. Erlotinib is a small molecule protein-kinase inhibitor targeting the epidermal growth factor receptor (EGFR) that may benefit a select subtype of cancer patients. However, identification of the characteristics of patients who may benefit from the use of erlotinib is not easy. Previous publication explored the activation of signal transduction proteins in the EGFR pathway as a method of determining erlotinib response and demonstrated that lung cancer cell lines characterized by increased pEGFR/pAKT ratios are more resistant to erlotinib. The aim of this study was to compare the activation of signal transduction markers in the EGFR pathway between immortalized cell lines and primary cultures of patient-derived tumor cells. Predictive potential of pEGFR/pAKT ratios in both immortalized cell lines and primary cultures of lung cancer cells was also compared.

Methods: Western blotting and In-Cell western analysis were used to determine the activation state of the EGFR signaling pathway. Signaling pathway components were compared in the presence and absence of erlotinib treatment in immortalized lung cancer cell lines and primary cultures of lung cancer specimens. Chemosensitivity of cell lines and specimens to erlotinib was determined using an in vitro drug response marker. Cells were treated with a 10 dose concentration range of erlotinib for 72 hours and the remaining post-treatment live cells were enumerated by nuclear staining.

Results: The pEGFR/pAKT ratio was increased in erlotinib-responsive immortalized cell lines when compared to non-responsive cell lines (p<0.05). This difference in pEGFR/pAKT ratio was significant after treatment with erlotinib as well. The difference in pEGFR/pAKT ratios in response to erlotinib treatment between erlotinib-responsive and non-responsive primary cultures of lung cancer cells was not, however, statistically significant.

Conclusion: These studies confirmed that pEGFR/pAKT ratios may be indicative of erlotinib response in immortalized lung cancer cell lines. However, when evaluating the signal transduction markers in patient-derived primary cultures, no correlation with in vitro erlotinib response was found. These results highlight the value of using in vitro drug response markers, which utilize primary patient tissue, in the development of predictive biomarkers.
Helicobacter pylori strain genotyping based on the amplification and sequencing of cagA gene
Authors: Garcia Daniella , Delgado Maria del Pilar , Quiroga Andres , Bravo Maria Mercedes , Jaramillo Carlos A.
Published: May 2015

Relevance: H. pylori presence has been described as a high risk factor of suffering gastric cancer, which is the fourth most common cancer in Colombia. Some studies [1, 2]; point out that when a genotypic characterization of different strains of this microorganism is carried out, more than one genotype can be found in the same patient can be found.

Purpose: To characterize different H.pylori cagA genotypes that occur in the same patient sample (biopsy) applying molecular and bioinformatic techniques.

Methods: cagA positive strains (n=35) from Instituto Nacional de Cancerología (Bogotá-Colombia), bank s samples were included as study material. Tissue DNA was extracted and used to amplify the 3’ region of the cagA gene by PCR [3]. The NTC11637 H. pylori strain was included as a positive control. PCR products were visualized in 2% agarose gels and the amplified fragments were purified using a commercial kit. As a final stage, a precipitation based on ethanol and EDTA was done and reaction products were sequenced. The obtained sequences were edited and submitted to a bioinformatic analysis using AASA, a program designed to characterize the EPIYA phosphorylation motifs localized in the C-terminal of the CagA protein by Rothstain, Yañez and Vivas (Los Andes University Thesis, 2008).

Results: The molecular and bioinformatic approach described allowed strain characterization. The applied strategy leads to verify the presence of the A, B and C EPIYA motifs in different combinations. ABC group was the most frequent motif found (48%). These results match those of previous reports in Colombia, nevertheless it is important to remark that amplicons with different molecular sizes can have the same EPIYA motif and that amplicons with similar sizes can have different EPIYA motifs.

Conclusions: The current strategy demonstrated to be useful in the identification and posterior characterization of more than one genotype of the different H. pylori strains present in the same biopsy sample. This molecular and bioinformatics approach combined with other techniques seems to be a good alternative to establish if an infection by H. pylori is the result of a coinfection or a microevolution process.

References: 1. Aras, R.A., et al., Natural variation in populations of persistently colonizing bacteria affect human host cell phenotype. The Journal of infectious diseases, 2003. 188(4): p. 486-496. 2. Panayotopoulou, E., et al., Strategy to characterize the number and type of repeating EPIYA phosphorylation motifs in the carboxyl terminus of CagA protein in Helicobacter pylori clinical isolates?†. Journal of Clinical Microbiology, 2007. 45(2): p. 488-495. 3. Yamaoka, Y., et al., Relationship between the cagA 3'repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH. Gastroenterology, 1999. 117(2): p. 342-349.
Regulation of Crm1 expression in cancer cells
Authors: Watt Pauline J. van der , Leaner Virna D.
Published: May 2015

Crm1 (or exportin 1) is a member of the karyopherin β protein family that mediates the nuclear export of cargo proteins and certain RNAs from the nucleus into the cytoplasm. We previously identified increased expression of Crm1 mRNA and protein in cervical tumours compared to normal epithelium, and increased Crm1 expression in transformed fibroblasts compared to normal fibroblasts. Moreover, Crm1 overexpression in cancer/transformed cells was found to be necessary for cell survival, implicating Crm1 as an attractive target for cancer therapy. We hypothesized that the increased expression of Crm1 in cancer/transformed cells derives from altered Crm1 promoter activity, yet no studies to date have investigated what regulates the Crm1 promoter. This study therefore set out to clone and characterize the Crm1 promoter. The cloned Crm1 promoter (-1405 to +99) was significantly more active in cancer and transformed cells compared to normal, correlating well with endogenous Crm1 protein levels. In order to define promoter regions responsible for the differential activity, deletion constructs were generated, and the −175 to +99 region was identified as important. Mutation of two CCAAT boxes and a GC box in this region significantly diminished Crm1 promoter activity in transformed and cancer cells, while having only a minor effect on Crm1 promoter activity in normal cells. In addition, ChIP assays revealed in vivo binding of NFY/CBP and Sp1 transcription factors to the CCAAT boxes and GC box of the Crm1 promoter in cancer and transformed cells, while binding was undetectable in normal cells. Interestingly, NFY and Sp1 expression levels were considerably elevated in transformed and cancer cells compared to normal. Taken together, we show that NFY- and Sp1-mediated activation of the Crm1 promoter contributes to Crm1 overexpression in cancer and transformed cells.
The role of p21-activated kinase 3 (PAK3) in activating protein 1 (AP-1) induced oncogenesis
Authors: Holderness Nina , Donninger Howard , Birrer Michael J. , Leaner Virna
Published: May 2015

Activating protein 1 (AP-1) is a fundamental transcription factor that plays a role in cell proliferation, differentiation and apoptosis. Little is known about the role of the diverse downstream targets of the active AP-1 complex, a dimer comprising of the Jun, Fos or ATF bZIP-domain proteins. Deregulation of AP-1 has been linked to many cancers, with its over-expression leading to the transformation of normal cells. Previous studies utilizing a doxycycline-inducible cJun/AP-1 construct identified PAK3, a serine/threonine kinase signal transduction molecule, as a potential AP-1-target involved in the AP-1 characteristic transformation. PAK3 has been implicated in a variety of pathological disorders and over-expression of other PAK-family members has been linked to certain cancers. This project aims to investigate the role of PAK3 in cJun/AP-1 induced oncogenesis. Quantitative RT-PCR and Western Blot Analysis showed elevated PAK3 expression at both the mRNA and protein level in cJun-overexpressing cells. PAK3 expression levels were also elevated in transformed human and cancer cell lines compared to normal cells. Analysis of the PAK3 promoter using promoter luciferase assays identified a putative AP-1 binding site, at position +52 to +60, which may be responsible for the increased PAK3 promoter activity in response to cJun/AP-1 over-expression. siRNA inhibition of the PAK3 protein showed the regression of the morphological phenotype and migratory potential associated with cJun-transformed cells. However PAK3 inhibition showed no significant effect on the proliferative response of cJun-transformed cells. This study suggests a potential regulation of cJun/AP-1 on PAK3 as well as a role for PAK3 in AP-1 induced transformation; specifically that associated with cell morphology and migration.
Dysregulation of the expression of miR204 mediates migration and invasion of endometrial cancer by regulating FOXC1
Authors: Wong Y. F. , Cheung T.H. , Lau T.S. , Chan L.K.Y. , Yim S.F. , Wang V.W. , Smith D.I.
Published: May 2015

microRNAs (miRNAs) regulate gene expression and some have been shown to function as either oncogenes or tumor suppressors (oncomirs). Changes in miRNA expression have been observed in many human cancers but their role in endometrioid endometrial cancer (EEC) remains poorly understood. We studied the expression of 365 human miRNAs using Taqman low density arrays in 98 EECs as well as 60 normal endometria. Potentially interesting miRNAs were then validated by qRT-PCR.

Expression of highly deregulated miRNAs on malignant cell properties was also examined in vitro using pre-/anti-miRNA transfection. Predicted mRNA targets for the miRNA of interest were also verified. We identified 16 significantly deregulated miRNAs in EEC and 7 of these are reported for the first time. Of these, miR-7, miR-194 and miR-449b knockdown and miR-204 induction repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. For the cancer progression dysregulation of the expression of miR204 mediates migration and invasion of endometrial cancer by regulating FOXC1-related miR-204, FOXC1 was determined as its target gene possessing two miR-204-binding sites at the 3’-untranslated region. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis also revealed the involvement of FOXC1 in migration of HEC1A cells.

We have identified dysfunctional miRNAs in endometrial cancer and elucidated the crucial role of miR-204-FOXC1 interaction in endometrial cancer progression. This work provides insight into miRNA-based diagnostic and therapeutic approaches for endometrial cancers.
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: May 2015

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 90-95% 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 semi quantitative RT-PCR was done, which showed markedly reduced expression of ALP mRNA in RCC as compared to adjacent normal kidney.
Trastuzamab antibody drug conjugates demonstrate robust in vitro antitumor activity in HER2 amplified breast cancer cell lines
Authors: Mohan Sankar , O'Brien Carol , Savage Heidi , Phillips Gail , Stern Howard , Lackner Mark , Pandita Ajay
Published: May 2015

Breast cancer is a heterogeneous disease with molecular subtypes characterized by distinct histopathology and gene expression signatures. HER2 oncogene amplification and over-expression is a defining feature of the HER2-amplified subtype of breast cancer. Currently, assays for detecting gene amplification and protein over-expression are used as diagnostic assays for the selection of patients for HER2-targeted therapies. In the present study, we compared the sensitivity of several HER2-targeted therapies across a panel of 31 breast cancer cell lines and correlated the sensitivity of the cell lines with HER2 positivity, as determined by IHC, qPCR and FISH. The agents utilized for the cell line screening were trastuzamab (Herceptin), two trastuzamab antibody-drug conjugates (ADCs) that employ an anti-mitotic agent, and a small molecule inhibitor of HER2 kinase activity. HER2 testing revealed 32% of the cell lines to be positive for HER2 by qPCR and FISH, and 90% of these demonstrated gene amplification in the form of clusters, as detected by FISH, or more than 10 copies as detected by qPCR. Several of the cell lines exhibited polysomy for chromosome 17 with accompanying extra copies of HER2 but did not meet the criteria for HER2 amplification. The HER2 IHC assay revealed 10 cell lines to be positive (2+, 3+) by IHC, with 8 exhibiting high-level expression (3+). The agents tested were more efficacious in the HER2-amplified cell lines, with wideranging activity observed in response to the ADCs. Cell lines exhibiting polysomy for HER2 and 1+ expression by IHC were less responsive to any of the agents tested. Consistent with previous reports, many of the cell lines resistant to trastuzamab or the small molecule HER2 inhibitor exhibited deregulation of the PI-3 kinase pathway illustrated by mutations in the PIK3CA gene or loss of PTEN protein expression. Interestingly, the majority of these resistant cell lines were sensitive to ADCs. In summary, our study demonstrates a high sensitivity to ADCs in breast cancer cell lines harboring high-level amplification of the HER2 gene and suggests these agents may overcome in vitro resistance conferred by activation of the PI-3 kinase pathway.
A simple method based on fluorescence probes for cancer diagnosis
Authors: Chang Ta-Chau , Kang Chi-Chih
Published: May 2015

A simple, rapid, and noninvasive method at low cost based on fluorescence of 3,6-Bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) staining was applied for point-of-care screening of cancer cells [1]. Further application to clinic malignant neck lumps showed that the diagnostic accuracy of this simple method alone is ~80 % [2]. In addition, the combination of the BMVC test and the fine-needle aspiration (FNA) cytology can reduce the non-diagnostic cases and improve the diagnostic accuracy. Since a small amount of the sample collected for clinic test is used for BMVC test, it can be widely used in Hospital and Medical Center either for a first-line screening tool or an adjuvant method to other methods for cancer diagnosis.

The success of the BMVC test for cancer diagnosis is mainly due to two distinct properties of BMVC: significant increase of the fluorescence quantum yield upon interaction with DNA and dramatic difference in the intracellular localization between the nucleus of cancer cells and the lysosome of normal cells. Here we have modified the molecular properties of BMVC derivatives to elucidate the possible correlation between molecular structure and cellular localization. Moreover, a better fluorescence probe than BMVC is screened for the detection of a single cancer cell by optimizing fluorescence contrast between cancer cells and normal cells by using a simple device. Our results not only provide invaluable information for targeting specific cellular organelles, but also establish new molecular principles for cancer diagnosis and treatment design. Further experiments show that BMVC can kill the CL1-0 cancer cells, but less damage the MRC-5 normal cells after irradiation at ~480 nm for several seconds.

1. Kang, C.C. et al. Analyst 132, 745-9 (2007).

2. Liao, L.J. et al. Analyst 134, 708-11 (2009).
Developing novel peptide inhibitors to chromosomal cohesion using mRNA display for nanodrug delivery in breast cancer therapy
Authors: McCullum Elizabeth O. , Pati Debananda
Published: May 2015

Several specific molecular networks mediate the cellular proliferation of tumorigenic cells. One signaling pathway of interest is the transition from metaphase to anaphase which is dependent on cleavage of the cohesin protein complex by separase, an endopeptidase. A normal transition allows the protein complex to form, sister chromatid cleavage and the onset of anaphase. However, failure to properly assemble or maintain the integrity of this complex results in the premature separation of chromatids (PCS), mitotic arrest and apoptosis in cultured cells. This research effort will focus on the identification of cohesin Rad21, a proapoptotic protein that is over-expressed in breast cancer cells, mimcs for the induction of apoptosis in the cohesin-disrupted tumors. Using mRNA display, Rad21 mimetopes will be selected to bind a SMC-1 target in the formation of standard cohesin complex assembly. The pool of isolated peptides will be further characterized for their specificity, induction of PCS, mitotic arrest and apoptosis both in vitro and in vivo. Finally, peptides will be conjugated to a RAFT-peptide scaffold drug delivery system to exclusively target rapidly proliferating breast tumor cells. The potential advantages provide an area of promising therapeutics for antitumor effects when coupled with chemotherapy at the point of early detection.
Growth control of human ovarian cancer cells by the dynein light chain and TGFβ receptor interacting protein km23-1
Authors: Mulder Kathleen M. , Pulipati Nageswara Rao , Liu Xin , Sun Baodong , Zhao Yan , Jin Qunyan , Ding Wei , Pandey Manoj K. , Huber Jonathan P.
Published: May 2015

We have identified km23-1 as a TGFβ signaling intermediate that is also a light chain of the minus-end directed motor protein dynein (DLC). Our results suggest that km23-1 (also termed DYNLRB1/robl1/mLC7-1/Dnlc2a) can function as a “motor receptor” to recruit TGFβ signaling components to the dynein motor for endosomal trafficking prior to nuclear translocation. For example, we have shown that overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocks TGFβ-stimulated nuclear translocation of Smad2. Further, siRNA blockade of km23-1 resulted in a reduction both in nuclear levels of phosphorylated (ie, activated) Smad2 and of TGFβ/Smad2-dependent transcriptional activity (JBC 282: 19122, 2007). Similarly, we have shown that km23-2 is required for Smad3 TGFβ signaling (JCP 221: 707, 2009). However, many human ovarian cancer cells (HOCCs) have lost growth inhibitory responsiveness to TGFβ. In order to assess whether km23-1 could also regulate the growth of aggressive, TGFβ-resistant HOCCs, we developed a tet-off-inducible model system for induction of wild-type km23-1 expression in SKOV-3 HOCCs. Here we show that overexpression of km23-1 (by 10-20 fold) suppressed the growth of the cells in vitro and markedly impeded tumor formation in vivo. Fluorescence-activated cell sorting (FACS) analysis in the SKOV-3 model system demonstrated an increased fraction of cells in G2/M after induction of km23-1 expression (dox removal), compared with controls. Similar results were obtained for the TGFβ-resistant IGROV-1 HOCCs. Additional studies revealed that km23-1 overexpression in HOCCs increased the mitotic index, in association with activation of both MEK and ERK and an elevation of cyclin B1 protein expression levels. The elevated levels of phospho-MEK, phospho-ERK, and cyclin B1, as well as the distribution of the cells in mitosis, suggest that the cells are delayed in prometaphase/ metaphase. This mitotic delay, followed by cell death, appears to be the mechanism by which high levels of km23-1 can suppress ovary tumor growth in vivo. Since dynein is known to have critical functions at this point in mitosis by inactivating the spindle assembly checkpoint (SAC), studies are underway to investigate how km23-1 overexpression regulates the SAC to cause the reduced tumor formation in vivo. This work was supported by NIH grants CA100239, CA92889, and CA90765 to KMM. Our results indicate that km23-1 is a critical ovarian cancer target for the development of novel diagnostic and therapeutic agents. In this regard, US patents have been issued to KMM related to km23-1-based methods for diagnostic/prognostic screening, as well as for the identification of km23-1-based therapeutic agents for ovarian cancer (US 7,229,758 B2; 6-07 & US 7,741,043; 6-10).
Phosphoproteomics and mass-action modeling to predict optimal drug combination in signaling networks
Authors: Morales Fabiana C. , Iadevaia Sergio , Lu Yiling , Ram Prahlad , Mills Gordon B.
Published: May 2015

Targeted therapeutics hold tremendous promise in inhibiting cancer cell proliferation. However, targeting proteins individually can be compensated for by bypass mechanisms and activation of regulatory loops. Designing optimal therapeutic combination must therefore take into consideration the complex dynamic networks in the cell. In this study, we analyzed the insulinlike growth factor (IGF-1) signaling network in the MDA-MB231 breast cancer cell line using reverse phase protein arrays to quantify the dynamic crosstalk between the phosphoinositide 3-kinase (PI3K), the mitogen-activated protein kinase (MAPK) and the mammalian target of rapamycin (mTOR) pathways. We developed a computational procedure that integrated mass-action modeling with particle swarm optimization to train the model against the experimental data and infer the unknown model parameters. The trained model was used to predict how targeting individual signaling proteins altered the rest of the network and identify drug combination that minimally increased phosphorylation of other proteins elsewhere in the network. Experimental testing of the modeling predictions showed that optimal drug combination inhibited cell signaling and proliferation, while non-optimal combination of inhibitors increased phosphorylation of non-targeted proteins and rescued cells from cell death. The integrative approach described here is useful for generating experimental intervention strategies that could optimize drug combination and discover novel pharmacologic targets for cancer therapy.
Affinity and size capture of circulating tumor cells: A platform for increased sensitivity
Authors: Maimonis Peter J. , Merdek Keith , Dietenhofer Kurt , Yen Lucy , Palmer Gary
Published: May 2015

Background: The potential clinical value of evaluating circulating tumor cells (CTCs) from blood is clear. CTC analysis can aid in cancer diagnosis, prognosis, and treatment prediction. However, since CTCs are rare, current analysis is limited by low efficiencies. Variability of expression of cell surface antigens by CTCs make capture mechanisms based on antibody affinity less than ideal. We report here data from a CTC platform that combines affinity capture with size filtration capture.

Methods: A spiked cell model was used, involving whole EDTA blood spiked with SKBR-3 human breast cancer cells. Two microfluidic chips were analyzed, a standard chip (T7) with constant gap size between posts, and a gradient chip (MA3) with a gap-size gradient between posts starting at 40 μ and decreasing to 12 μ. Chips were coated with either an anti-EpCAM monoclonal antibody or with no Ab. An anti-cytokeratin (CK) Ab in combination with a nuclear specific fluorescent stain on an automated cell imaging platform was used to identify CTCs.

Results: 45% of cells were captured by size alone (MA3 with no Ab), 16% of cells were captured by affinity alone (T7 with MAb), and 65% of cells were captured by a combination of antibody affinity and size gradient (MA3 with MAb). This increased capture efficiency has been confirmed in clinical samples from patients with metastatic breast cancer, with a greater than 40% increase in capture efficiency utilizing both mechanisms. Whole blood from healthy donors run through either chip type yielded virtually no CTCs. FACS analysis showed that 75% of cultured SKBR-3 cells express high levels of EpCAM. This expression level will vary, as will, presumably, the relative contributions of the antibody capture mechanism and the size capture mechanism. A “heat map” analysis can demonstrate visually the relative contributions of each and these will be presented.

Conclusions: The MA3 microfluidic chip captures CTCs using a combination of affinity and size filtration. This allows for the capture of CTCs with varying expression levels of EpCAM by exploiting the size differential between these CTCs and other blood elements. This dual-capture mechanism may allow for more efficient capture than affinity alone. Further work evaluating this platform in late-stage breast cancer patients is ongoing.
Aberrant methylation in breast cancer and correlate with tumor phenotypes, prognostic factors, and patient outcome
Authors: Martinez-Galan Joaquina , Delgado-Perez Juan Ramón , Torres-Torres Blanca , Ballesteros Pedro , Gonzalez-Flores Encarnación , Valdivia Javier , Luque Raquel , Conde Veronica , Ortega Jose Antonio , de Almodovar Mariano Ruiz
Published: May 2015

Purpose: Posttranslational modifications are known to be altered in cancer cells, and loss of selected histone acetylation and methylation marks has recently been shown to predict patient outcome in human carcinoma.

Materials: Since January 2002 to June 2005, 107 women with breast cancer and 108 control subjects were recruited. A sensitive SYBR green methylation-specific PCR quantitative technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of APC, RAR-β, E-cadherin, ESR1 and 14-3-3σ gene promoter regions as breast cancer biomarkers. Sera were collected in 107 operable breast cancer patients (pts) previously surgery and in 60 of those pts after treatment. Respect controls, 34 had benign breast disease and 74 with no evidence of breast disease

Results: Mean serum values of methylated ESR1 and 14-3-3σ gene promoters significantly differed between breast cancer patients and healthy controls (p = 0.0112 for ESR1 and p= 0.0047 for 14-3-3- σ). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (p≤0.0001) with a sensitivity of 81% (95% confidence interval: 72—88%) and specificity of 88% (95% CI: 78—94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p = 0.0179); and presence of methylated 14-3-3-σ was associated with T3-4 stage (OMS) (p< 0.05) and nodal positive status (p< 0.05). We observed lower methylated ERS1 or 14-3-3-σ values after surgery, respect pretreatment levels, but without an overall statistically significant difference. With a median follow up of 6 years, we found that patients with a significant decrease of sera methylated levels of both genes after surgery had better time to progression an overall survival respect patients without this observation

Conclusions: These results also suggest that this panel of genes detected in ductal lavage and blood specimens could be useful to biomarkers for early detection breast cancer. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA is a promising tool for cancer risk assessment.
Nanotechnology and individualized oncology
Authors: Ferrari Mauro
Published: May 2015

The advent of novel engineering technologies affords unprecedented advances toward long-elusive objectives of medical research. Individualized medicine responds to the basic but generally unattainable question of identifying the right therapy, reaching the right therapeutic target in the body at the right time, and securing immediate feedback as for its efficacy and undesired collateral effect. Finally, individualized medicine appears to be a credible general objective in cancer, owing to the integration of classical disciplines of clinical medicine, methods of molecular biology, and novel technology platforms.

Nanotechnologies are of great interest in the context of the drive toward individualized oncology, and may prove to be the necessary catalyst for its large-scale implementation. In this talk I will present nanoporous-silicon-based approaches for the individualization of medical intervention: multistage vectors for the preferential localization of therapeutic agents; therapeutic monitoring nanotextured chips for the proteomic and peptidomic content profiling of biological samples; nanochannel delivery systems for intelligent time-release from implants, and bionanoscaffolds for post-traumatic osteoregeneration.

While novel nanoplatforms engender direct clinical applications, at the same time they afford the formulation of novel frameworks and hypotheses for the basic understanding of pathological processes. In particular, multistage particulates are the probes that afford the exploration of a new perspective of cancer, that is, that the unifying aspect of the canonical ‘hallmarks of cancers’ all relate to dysregulation of mass transport at scales including the molecular, cellular, microenvironmental, and systemic. These considerations are the starting point for “Transport OncoPhysics.”
Clinical trials using novel molecular imaging cancer biomarkers: Current status and future directions
Authors: Mankoff David A.
Published: May 2015

The ability to assay cancer biologic features is key to advances in the both the basic biomedical science and clinical patient care. Advances in our ability to assay molecular processes including gene expression, protein expression, and molecular and cellular biochemistry have fueled recent advances in our understanding of many diseases and our ability to treat them. Most assays require sampling of cells or tissue to perform the measurements. Thus the cell culture system, animal model, or patient must be perturbed in order to perform the assay. This requirement makes serial assays over time more difficult and leaves open the possibility that the assay itself may change the state of the system being measured. The ability to measure biologic processes without perturbing them would be highly desirable and would offer complementary information to that obtained by most traditional assay methods. The ability to image regional biochemistry and molecular biology, termed molecular imaging [1,2] offers great promise in using molecular imaging as a cancer biomarker. Quantitative capability is an important feature of molecular imaging, for example, the ability to measure regional tumor receptor expression. The quantitative and non-invasive nature of molecular imaging makes it a potentially very powerful tool for cancer drug development and testing and for translational science [3].

This talk will review the use of molecular imaging as a biomarker, particular positron emission tomography (PET). Examples will be shown using PET imaging to (1) provide prognostic information based upon in vivo measures of cancer differentiation and aggressiveness, (2) measure the regional expression of possible therapeutic targets, (3) provide early assessment of response to therapy. Current clinical trials involving PET as a cancer biomarker will be reviewed, including those using PET radiopharmaceuticals beyond 18F-fluoridexoyglucose (FDG) [3, 4]. The spectrum of current trials will be discussed, with an emphasis on current NCI and ACRIN clinical trials of novel imaging probes, and an eye towards matching future trials of targeted cancer therapy with companion molecular imaging trials.
Cancer genomics and the TCGA project
Authors: Haussler David
Published: May 2015

Large-scale cancer genomics projects such as The Cancer Genome Atlas (TCGA) and the International Cancer Genomics Consortium (ICGC) will sequence tens of thousands of tumors in the new few years, along with matched normal blood samples and other normal tissues. TCGA alone plans to analyze 500 clinically characterized samples from each of 20 different cancer types, detecting frequently mutated genes, common copy number variants, altered gene expression and methylation changes. These data will provide an exceptional resource for identifying new diagnostic targets and predictors of response.

Building on technology developed for the UCSC genome browser, we have developed a cancer genome analysis pipeline and a cancer genomics browser to interpret cancer genomics data that will aid in the identification of new targets. We use a new approach called PARADIGM, based on factor graphs, to map multiple data types into a single coherent pathway model for higher level interpretation. PARADIGM currently employs models for many hundreds of pathways from pathway libraries such as NCI pathways, Wikipathways, KEGG, etc. By transforming raw genomic data to pathway activity levels, PARADIGM provides a comprehensible window into the data that can be coupled to predictors of response to improve accuracy. We discuss the early results of this approach to the TCGA data.
Prediction of disease risk using common genetic variants
Authors: Hunter David J.
Published: May 2015

A frequently proposed application of knowledge on inherited predisposition to cancers is the possibility that susceptible individuals could be targeted for enhanced surveillance, lifestyle advice, or chemoprevention, and thus have reduced risk of diagnosis or mortality from cancer. Action based on knowledge of mutations in high penetrance genes such as BRCA1 in breast cancer, or hMSH2 and colorectal cancer has become established practice in high risk clinics. With the advent of genome-wide association studies (GWAS) dozens of common genetic variants (mostly SNPs) have been discovered to be associated with cancer at various sites, with approximately 30 discovered for prostate cancer alone. In general, the relative risks associated with these risk variants are an order of magnitude or more less than those associated with high penetrance gene mutations, and thus, the risk information contributed by a single common risk variant contributes minimally to risk prediction. However, when cumulated into multi-SNP risk scores, large relative risks are predicted for the small number of persons in the tail of the risk distribution (i.e. those who have inherited mostly risk variants as opposed to “protective” variants). For this small number of people, the estimated relative risks approach those for the high penetrance single gene mutations, but at this stage of our knowledge the risk scores are unstable and may change as more common variants are discovered. For the majority of people, with either modestly elevated or modestly reduced predicted risks, the clinical application of this knowledge is even less secure. However, combinations of risk variants have been shown at some cancer sites to be at least as predictive as available risk scores based on demographic and lifestyle factors. As more variants are discovered, the predictive value of these risk scores will increase, and thus the clinical utility of the information should be rigorously tested.
Potential clinical utilities of prostate cancer risk-associated SNPs
Authors: Xu Jianfeng
Published: May 2015

Prostate cancer (PCa) is the most common solid organ malignancy affecting American men and the second leading cause of cancer related death. Recently, more than 30 PCa risk-associated single nucleotide polymorphisms (SNPs) have been discovered from genome-wide association studies (GWAS). These risk-associated SNPs have been consistently replicated in multiple case-control study populations of European descent. Although each of these SNPs is only moderately associated with PCa risk, a genetic score based on a combination of risk-associated SNPs can be used to identify men at high risk for PCa.

However, these results were obtained from case-control studies which have several significant limitations. The validity of these genetic associations, the relative performance of genetic markers for PCa risk prediction compared to existing clinical variables such as PSA, and more importantly, whether genetic markers add value to existing clinical variables, are key questions that need to be addressed in well-controlled prospective studies prior to their consideration for clinical use.

In this presentation, I will present the findings of PCa risk prediction using multiple risk-associated SNPs. I will also discuss our effort to assess clinical validity and clinical utility of these genetic markers in the Reduction by DUtasteride of Prostate Cancer Events (REDUCE) study, a large randomized clinical trial. All men in the REDUCE trial had a negative biopsy at baseline and were followed-up for four years, with scheduled not-for-cause (i.e. regardless of PSA levels and other clinical indications) prostate biopsies at years 2 and 4. Therefore, this study design minimizes the potential impact of PSA detection bias on associations between PCa risk and SNPs. In addition, because it is a clinical trial, a number of clinical variables, such as free/total PSA ratio and prostate volume were measured at baseline using a standardized protocol. Therefore, it allows us to compare performance of genetic markers and existing clinical variables in predicting positive prostate biopsy in the same study. Finally, the study design allows us to assess whether genomic-targeted chemoprevention, based on estimated risk from genetic markers, can improve efficacy of chemoprevention. This study represents an important next and critical step towards an assessment of the potential clinical utility of genetic markers.
Deep collective cancer invasion: Breaking radioresistance by anti-integrin therapy
Authors: Friedl Peter
Published: May 2015

The tumor microenvironment contributes to cancer invasion, growth and survival and thereby impacts tumor responses to therapy. We here show for orthotopic fibrosarcoma and melanoma xenografts deep invasive growth driven by proliferation concurrent with collective invasion of multicellular strands along the normoxic perivascular stroma. Invasion was fast (up to 200 µm per day), non-destructive and independent of β1 and β3 integrins. Despite normoxia, perivascular invasion strands were resistant to high-dose hypofractionated irradiation which otherwise was sufficient to induce regression of the tumor main mass. This invasion-associated radioresistance was sensitive to the simultaneous inhibition of β1 and β3 integrins by RNA interference or combined anti-β1/aV integrin antibody treatment caused byproliferation arrest, anoikis induction ablating both tumor lesion and invasion strands. Thus, collective invasion is an important invasion mode in solid tumors into a microenvironmentally privileged perivascular survival niche which conveys radioresistance by integrin-dependent signals. Consequently, combining anti-integrin therapy with hypofractionated irradiation may be amenable to clinical cancer treatment of locally destructive and otherwise radioresistant tumor lesions.
Angiogenesis success and pitfalls in diagnosis, therapy and cancer prevention
Authors: Albini Adriana
Published: May 2015

In contrast to conventional chemotherapy, which targets the genetically mutated cancer cells, anti-vascular tumor therapy is primarily directed towards the newly formed blood vessel compartment, and is supposed to have the some advantages compared to conventional cytotoxic chemotherapy: (I) it is not restricted to a certain histologic tumor type, since all solid tumors depend on angiogenesis and the maintenance of functional microvasculature; (II) the tumor microvasculature is well accessible to systemic treatment; (III) it reaches the whole tumor mass, since every single tumor microvessel has to supply up to hundreds of critically dependent tumor cell layers; (IV) angiogenesis in adult organisms is only induced during the reproductive ovarian cycle or wound healing, thus an antagonism of angiogenesis should be a highly selective therapy; (V) the endothelial cell is genetically stable and therefore less prone to development of drug resistance. Recent evidences, however, show recurrence of tumor growth after an initial period of suppression during long-term therapy using antiangiogenic agents. Redundancy of alternative proangiogenetic factors in antiangiogenic monotherapies, upregulation of proangiogenic or anti-apoptotic factors, or heterogeneity of vascular dependency among tumor subpopulations are thought to be possible mechanisms for this phenomenon, as well as vascular remodeling. On the other hand, targeting angiogenic pathways can undermine elements and function of the cardiovascular system leading to potentially deadly side effects ranging from hypertension to heart failure. It is important to reconsider all the implications of antiangiogenic treatment to improve the therapeutic outcome and patients’ well being.