CDCP1 (CUB domain containing protein 1)

Review on CDCP1 (CUB domain containing protein 1), with data on DNA, on the protein encoded, and where the gene is implicated

Quantifying the CDK inhibitor VMY-1-103\u27s activity and tissue levels in an in vivo tumor model by LC-MS/MS and by MRI.

The development of new small molecule-based therapeutic drugs requires accurate quantification of drug bioavailability, biological activity and treatment efficacy. Rapidly measuring these endpoints is often hampered by the lack of efficient assay platforms with high sensitivity and specificity. Using an in vivo model system, we report a simple and sensitive liquid chromatography-tandem mass spectrometry assay to quantify the bioavailability of a recently developed novel cyclin-dependent kinase inhibitor VMY-1-103, a purvalanol B-based analog whose biological activity is enhanced via dansylation. We developed a rapid organic phase extraction technique and validated wide and functional VMY-1-103 distribution in various mouse tissues, consistent with its enhanced potency previously observed in a variety of human cancer cell lines. More importantly, in vivo MRI and single voxel proton MR-Spectroscopy further established that VMY-1-103 inhibited disease progression and affected key metabolites in a mouse model of hedgehog-driven medulloblastoma

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer.

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab

The Structural Features of Trask That Mediate Its Anti-Adhesive Functions

Trask/CDCP1 is a transmembrane protein with a large extracellular and small intracellular domains. The intracellular domain (ICD) undergoes tyrosine phosphorylation by Src kinases during anchorage loss and, when phosphorylated, Trask functions to inhibit cell adhesion. The extracellular domain (ECD) undergoes proteolytic cleavage by serine proteases, although the functional significance of this remains unknown. There is conflicting evidence regarding whether it functions to signal the phosphorylation of the ICD. To better define the structural determinants that mediate the anti-adhesive functions of Trask, we generated a series of deletion mutants of Trask and expressed them in tet-inducible cell models to define the structural elements involved in cell adhesion signaling. We find that the ECD is dispensable for the phosphorylation of the ICD or for the inhibition of cell adhesion. The anti-adhesive functions of Trask are entirely embodied within its ICD and are specifically due to tyrosine phosphorylation of the ICD as this function is completely lost in a phosphorylation-defective tyrosine-phenylalanine mutant. Both full length and cleaved ECDs are fully capable of phosphorylation and undergo phosphorylation during anchorage loss and cleavage is not an upstream signal for ICD phosphorylation. These data establish that the anti-adhesive functions of Trask are mediated entirely through its tyrosine phosphorylation. It remains to be defined what role, if any, the Trask ECD plays in its adhesion functions

Optical fiber‐based in vivo quantification of growth factor receptors

BACKGROUND: Growth factor receptors such as epidermal growth factor receptor 1 and human epidermal growth receptor 2 (HER2) are overexpressed in certain cancer cells. Antibodies against these receptors (eg. cetuximab and transtuzumab [Herceptin]) have shown therapeutic value in cancer treatment. The existing methods for the quantification of these receptors in tumors involve immunohistochemistry or DNA quantification, both in extracted tissue samples. The goal of the study was to evaluate whether an optical fiber‐based technique can be used to quantify the expression of multiple growth factor receptors simultaneously. METHODS: The authors examined HER2 expression using the monoclonal antibody trastuzumab as a targeting ligand to test their system. They conjugated trastuzumab to 2 different Alexa Fluor dyes with different excitation and emission wavelengths. Two of the dye conjugates were subsequently injected intravenously into mice bearing HER2‐expressing subcutaneous tumors. An optical fiber was then inserted into the tumor through a 30‐gauge needle, and using a single laser beam as the excitation source, the fluorescence emitted by the 2 conjugates was identified and quantified by 2‐photon optical fiber fluorescence. RESULTS: The 2 conjugates bound to the HER2‐expressing tumor competitively in a receptor‐specific fashion, but they failed to bind to a similar cell tumor that did not express HER2. The concentration of the conjugate present in the tumor as determined by 2‐photon optical fiber fluorescence was shown to serve as an index of the HER2 expression levels. CONCLUSIONS: These studies offer a minimally invasive technique for the quantification of tumor receptors simultaneously. Cancer 2012;. © 2011 American Cancer Society. The paper describes the in vivo quantification of human epidermal growth receptor 2 using a minimally invasive 2‐photon optical fiber fluorescence detection technique. The proof of concept for the simultaneous in vivo quantification of multiple receptors is provided.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91221/1/26487_ftp.pd

HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells

BACKGROUND: The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses. METHODOLOGY AND PRINCIPAL FINDINGS: Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment-induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa. CONCLUSIONS AND SIGNIFICANCE: These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients

Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting

<p>Abstract</p> <p>Background</p> <p>Testicular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.</p> <p>Methods</p> <p>Human embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-<it>trans </it>retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.</p> <p>Results</p> <p>Canonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.</p> <p>Conclusion</p> <p>During induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.</p

Agreement between chromogenic in situ hybridisation (CISH) and FISH in the determination of HER2 status in breast cancer

Determination of the HER2/neu (HER2) status in breast carcinoma has become necessary for the selection of breast cancer patients for trastuzumab therapy. Amplification of the gene analysed by fluorescence in situ hybridisation (FISH) or overexpression of the protein determined by immunohistochemistry (IHC) are the two major methods to establish this status. A strong correlation has been previously demonstrated between these two methods. However, FISH is not always feasible in routine practice and weakly positive IHC tumours (2+) do not always correspond to a gene amplification. Our study was performed in order to evaluate the contribution of chromogenic in situ hybridisation (CISH), which enables detection of the gene copies through an immunoperoxidase reaction. CISH was performed in 79 breast carcinomas for which the HER2 status was previously determined by IHC and FISH. The results of IHC, FISH and CISH were compared for each tumour. CISH procedures were successful in 95% of our cases. Whatever the IHC results, we found a very good concordance (96%) between CISH and FISH. Our study confirms that CISH may be an alternative to FISH for the determination of the gene amplification status in 2+ tumours. Our results allow us to think that, in many laboratories, CISH may also be an excellent method to calibrate the IHC procedures or, as a quality control test, to check regularly that the IHC signal is in agreement with the gene statu

Comparing the Epidermal Growth Factor Interaction with Four Different Cell Lines: Intriguing Effects Imply Strong Dependency of Cellular Context

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of 125I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®