Morgana acts as a proto-oncogene through inhibition of a ROCK-PTEN pathway.

Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy.

Spontaneous and pronase-induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines.

A subgroup of HER2-overexpressing breast tumours co-expresses p95(HER2), a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95(HER2) expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2-positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95(HER2) expression. Immunohistochemistry was performed on parallel formalin-fixed, paraffin-embedded sections of the same case series using antibodies directed against either the intra- or extra-cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185(HER2)-positive/p95(HER2)-positive samples than in the p185(HER2)-positive/p95(HER2)-negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95(HER2)-positive cases. Conversely, the percentage of 2+ scored cells was higher inp95(HER2)-negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2-HER3 dimers was studied using a proximity-ligation assay. In vitro experiments showed that short-term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2-HER3 dimers, and did not interfere with pertuzumab-binding capacity. In conclusion, the presence of p95(HER2 as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab.

A New Anorganic Equine Bone Substitute for Oral Surgery: Structural Characterization and Regenerative Potential.

Different xenogeneic inorganic bone substitutes are currently used as bone grafting materials in oral and maxillo-facial surgery. The aim of the present study was to determine the physicochemical properties and the in vivo performance of an anorganic equine bone (AEB) substitute. AEB is manufactured by applying a process involving heating at >300 °C with the aim of removing all the antigens and the organic components. AEB was structurally characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray fluorescence (XRF), and Fourier-transformed infrared (FT-IR) spectroscopy and compared to the anorganic bovine bone (ABB). In order to provide a preliminary evaluation of the in vivo performance of AEB, 18 bone defects were prepared and grafted with AEB (nine sites), or ABB (nine sites) used as a control, in nine Yucatan Minipigs. De novo bone formation, residual bone substitute, as well as local inflammatory and tissue effects were histologically evaluated at 30 and 90 days after implantation. The structural characterization showed that the surface morphology, particle size, chemical composition, and crystalline structure of AEB were similar to cancellous human bone. The histological examination of AEB showed a comparable pattern of newly formed bone and residual biomaterial to that of ABB. Overall, the structural data and pre-clinical evidence reported in the present study suggests that AEB can be effectively used as bone grafting material in oral surgery procedures.

Current Challenges for HER2 Testing in Diagnostic Pathology: State of the Art and Controversial Issues.

HER2 overexpression and anti-HER2 agents represent probably the best story of success of individualized therapy in breast cancer. Due to the important therapeutic implications, the issue under the spotlight has been, since ever, the correct identification of true HER2 positivity on tissue specimens. Eligibility to anti-HER2 agents is strictly dependent on the demonstration of HER2 overexpression (by immunohistochemistry) or of HER2 gene amplification by in situ techniques (fluorescence in situ hybridization, FISH), however there are controversial issues involving cases with "equivocal" HER2 status based on conventional techniques (about 20% of specimens). In terms of HER2 expression a major debate is the presence of full-length and truncated forms of the protein and controversial clinical data have been reported on the therapeutic implications of these HER2 fragments. In terms of HER2 gene assessment, the occurrence of amplification of the chromosome 17 centromeric region (CEP17) has been proven responsible for misleading HER2 FISH results, precluding anti-HER2 based therapy to some patients. Finally HER2 activating mutations have been recently described as a biological mechanisms alternative to HER2 gene amplification. In this review we will focus on the controversies that pathologists and oncologists routinely face in the attempt to design the most tailored treatment for breast cancer patients. We will focus on the HER2 gene and on the protein, both at technical and interpretational levels.

Aberrant expression of TfR1/CD71 in thyroid carcinomas identifies a novel potential diagnostic marker and therapeutic target.

BACKGROUND: Type I receptor for transferrin (TfR1/CD71) is overexpressed in several malignant tumors, but no studies are available on thyroid carcinomas. Our previous comparative analyses of the relative distribution of transferrin in benign versus papillary thyroid carcinoma (PTC) tissues highlighted a marked malignancy-associated abundance of the molecule. The aim of the present study was to evaluate whether TfR1/CD71 is also differentially expressed in benign versus malignant thyroid tissues. METHODS: Tissue samples, including benign lesions and follicular-derived carcinomas, from 241 patients and a total of 35 benign and malignant fresh specimens were assayed for TfR1/CD71 expression by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. RESULTS: We found that transcription of TfR1/CD71 gene is constitutive in thyroid epithelia, but the mRNA is differently translated in benign and malignant tissues. Western blot revealed higher levels of TfR1/CD71 protein in malignant versus benign tissues. Immunohistochemically, most carcinomas exhibited overexpression of the receptor, predominantly in the cytoplasm of neoplastic cells. The highest expression level was detected in primary and metastatic papillary carcinomas and anaplastic carcinomas, with positive results ranging from 86% to 100% of the cases. In contrast, most benign tissues were negative, with only a minority of cases showing focal and weak immunoreactivity. CONCLUSIONS: Our findings suggest that altered expression of TfR1/CD71 may be used as a marker helpful in distinguishing PTC from papillary hyperplasia and follicular variant PTC from benign follicular-patterned lesions. Additionally, the present observations support the rationale for the use of radiolabeled transferrin/transferrin analogs and/or anti-TfR1/CD71 antibodies for diagnostic and/or radiotherapeutic purposes in TfR1/CD71-expressing thyroid tumors.