Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

The clinical significance of cathepsin S expression in human astrocytomas

Early local invasion by astrocytoma. cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-LeuHomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma. cells and provide evidence for a potential role for CatS in invasion.

Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels

The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.

Overexpression of neurone glial-related cell adhesion molecule is an independent predictor of poor prognosis in advanced colorectal cancer

A downstream target of the Wnt pathway, neurone glial-related cell adhesion molecule (Nr-CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr-CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr-CAM protein expression, compared to 10 / 245 (4%) matched normal tissue (P <0.0001). Of 428 CRC samples, 97 (23%) showed Nr-CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr-CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr-CAM overexpression correlated with a significant increase in disease-specific (HR 1.66; 95% confidence interval 1.11-2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07-2.30; P = 0.023) in advanced but not early stage disease. Notably, 5-fluorouracil-based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr-CAM overexpression but not to those with Nr-CAM overexpressed tumors. In conclusion, Nr-CAM protein expression is upregulated in CRC tissues. Nr-CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non-beneficence to 5-fluorouracil- based chemotherapy.

CCAAT/enhancer binding protein α predicts poorer prognosis and prevents energy starvation-induced cell death in hepatocellular carcinoma

CCAAT enhancer binding protein α (C/EBPα) plays an essential role in cellular differentiation, growth, and energy metabolism. Here, we investigate the correlation between C/EBPα and hepatocellular carcinoma (HCC) patient outcomes and how C/EBPα protects cells against energy starvation. Expression of C/EBPα protein was increased in the majority of HCCs examined (191 pairs) compared with adjacent nontumor liver tissues in HCC tissue microarrays. Its upregulation was correlated significantly with poorer overall patient survival in both Kaplan-Meier survival (P = 0.017) and multivariate Cox regression (P = 0.028) analyses. Stable C/EBPα-silenced cells failed to establish xenograft tumors in nude mice due to extensive necrosis, consistent with increased necrosis in human C/EBPα-deficient HCC nodules. Expression of C/EBPα protected HCC cells in vitro from glucose and glutamine starvation-induced cell death through autophagy-involved lipid catabolism. Firstly, C/EBPα promoted lipid catabolism during starvation, while inhibition of fatty acid beta-oxidation significantly sensitized cell death. Secondly, autophagy was activated in C/EBPα-expressing cells, and the inhibition of autophagy by ATG7 knockdown or chloroquine treatment attenuated lipid catabolism and subsequently sensitized cell death. Finally, we identified TMEM166 as a key player in C/EBPα-mediated autophagy induction and protection against starvation.

CONCLUSION: The C/EBPα gene is important in that it links HCC carcinogenesis to autophagy-mediated lipid metabolism and resistance to energy starvation; its expression in HCC predicts poorer patient prognosis.

Oncogenic Kras Promotes Chemotherapy-Induced Growth Factor Shedding via ADAM17.

Oncogenic mutations in Kras occur in 40% to 45% of patients with advanced colorectal cancer (CRC). We have previously shown that chemotherapy acutely activates ADAM17, resulting in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. In this study, we examined the role of mutant Kras in regulating growth factor shedding and ADAM17 activity, using isogenic Kras mutant (MT) and wild-type (WT) HCT116 CRC cells. Significantly higher levels of TGF-a and VEGF were shed from KrasMT HCT116 cells, both basally and following chemotherapy treatment, and this correlated with increased pErk (phosphorylated extracellular signal regulated kinase)1/2 levels and ADAM17 activity. Inhibition of Kras, MEK (MAP/ERK kinase)1/2, or Erk1/2 inhibition abrogated chemotherapy-induced ADAM17 activity and TGF-a shedding. Moreover, we found that these effects were not drug or cell line specific. In addition, MEK1/2 inhibition in KrasMT xenografts resulted in significant decreases in ADAM17 activity and growth factor shedding in vivo, which correlated with dramatically attenuated tumor growth. Furthermore, we found that MEK1/2 inhibition significantly induced apoptosis both alone and when combined with chemotherapy in KrasMT cells. Importantly, we found that sensitivity to MEK1/2 inhibition was ADAM17 dependent in vitro and in vivo. Collectively, our findings indicate that oncogenic Kras regulates ADAM17 activity and thereby growth factor ligand shedding in a MEK1/2/Erk1/2-dependent manner and that KrasMT CRC tumors are vulnerable to MEK1/2 inhibitors, at least in part, due to their dependency on ADAM17 activity.

Denaturing high performance liquid chromatography for the detection of microsatellite instability using Bethesda and pentaplex marker panels

Microsatellite instability (MSI) is a characteristic molecular phenotype of tumors from the hereditary nonpolyposis colorectal cancer (Lynch) syndrome. Routine MSI screening of tumors in patients is an efficient prescreening tool for the population-based detection of Lynch syndrome in the absence of family cancer history. We describe here the optimization of a denaturing high performance liquid chromatography (DHPLC) assay for MSI analysis with the

Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

BRCA1 Deficiency Exacerbates Estrogen-Induced DNA Damage and Genomic Instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptora- negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability.We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types. © 2014 American Association for Cancer Research.

Roadmap to clinical use of gold nanoparticles for radiation sensitization

The past decade has seen a dramatic increase in interest in the use of gold nanoparticles (GNPs) as radiation sensitizers for radiation therapy. This interest was initially driven by their strong absorption of ionizing radiation and the resulting ability to increase dose deposited within target volumes even at relatively low concentrations. These early observations are supported by extensive experimental validation, showing GNPs' efficacy at sensitizing tumors in both in vitro and in vivo systems to a range of types of ionizing radiation, including kilovoltage and megavoltage X rays as well as charged particles. Despite this experimental validation, there has been limited translation of GNP-mediated radiation sensitization to a clinical setting. One of the key challenges in this area is the wide range of experimental systems that have been investigated, spanning a range of particle sizes, shapes, and preparations. As a result, mechanisms of uptake and radiation sensitization have remained difficult to clearly identify. This has proven a significant impediment to the identification of optimal GNP formulations which strike a balance among their radiation sensitizing properties, their specificity to the tumors, their biocompatibility, and their imageability in vivo. This white paper reviews the current state of knowledge in each of the areas concerning the use of GNPs as radiosensitizers, and outlines the steps which will be required to advance GNP-enhanced radiation therapy from their current pre-clinical setting to clinical trials and eventual routine usage.

Potential role of intensity-modulated radiotherapy in the treatment of tumors of the maxillary sinus.

To assess 3-dimensional conformal radiotherapy (3D-CRT) and intensity-modulated radiotherapy (IMRT) techniques to see whether doses to critical structures could be reduced while maintaining planning target volume (PTV) coverage in patients receiving conventional radiotherapy (RT) for carcinoma of the maxillary sinus because of the risk of radiation-induced complications, particularly visual loss. Six patients who had recently received conventional RT for carcinoma of the maxillary sinus were studied. Conventional RT, 3D-CRT, and step-and-shoot IMRT plans were prepared using the same 2-field arrangement. The effect of reducing the number of segments in the IMRT beams was investigated. 3D-CRT and IMRT reduced the brain and ipsilateral parotid gland doses compared with the conventional plans. IMRT reduced doses to both optic nerves; for the contralateral optic nerve, 15-segment IMRT plans delivered an average maximal dose of 56.4 Gy (range 53.9–59.3) compared with 65.7 Gy (range 65.3–65.9) and 64.2 Gy (range 61.4–65.6) for conventional RT and 3D-CRT, respectively. IMRT also gave improved PTV homogeneity and improved coverage, with an average of 8.5% (range 7.0–11.7%) of the volume receiving