Activated Leukocyte Cell Adhesion Molecule Expression and Shedding in Thyroid Tumors

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology

Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line

BACKGROUND: TPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1. RESULTS: Dasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130(CAS), Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin β1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed. CONCLUSIONS: All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC

The Ret(C620R) Mutation Affects Renal and Enteric Development in a Mouse Model of Hirschsprung’s Disease

In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung’s disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RET(C620R) substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although Ret(C620R) homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. Ret(C620R) heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The Ret(C620R) allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease

Identification of MET and SRC Activation in Melanoma Cell Lines Showing Primary Resistance to PLX403212

PLX4032/vemurafenib is a first-in-class small-molecule BRAFV600E inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies

Release of ALCAM is sensitive to ADAM17/TACE silencing.

<p>(A) Analysis of lysates from TPC-1 and A2774 cells, resolved on 4–12% SDS-PAGE and immunoblotted with anti-ADAM17/TACE antibodies. Arrows indicate inactive (130 kDa) and active (80 kDa) forms of ADAM17/TACE enzyme. Normalization of results was obtained with immunoblotting analysis of beta-actin. (B) Expression of ADAM17/TACE protein by TPC-1 cells transfected with an ADAM17/TACE-specific small interfering RNA (siRNA) (OTP17), or with non-targeting siRNA (NT) as detected by western blot. The amount of protein was calculated by comparative densitometric scanning with beta-actin. (C) ELISA detection of ALCAM release by TPC-1 cells after transfection with siRNA specifically inhibiting ADAM-17/TACE (OTP17, black column) or with non-targeting siRNA (NT, white column). (D) Conditioned medium (CM) from TPC-1 cells, cultured with pervanadate (PV) (60 min), epidermal growth factor (EGF) (24 h), or medium alone (ctr, 24 h) in the presence (black columns) or in absence (white columns) of CGS27023A (CGS) was assessed by ELISA for ALCAM. Columns, means of three experiments (cells cultured in presence of 10, 1, or 0.1 µM CGS); bars, SD. *, P<0.05. Grey bar: in absence of CGS, but in presence of orthovanadate (OV).</p

ALCAM expression in thyroid cell lines.

<p>Analysis of TT and MZ-CRC1 cell lysates showing ALCAM protein expression in these thyroid cell lines. SKOV-3 and TPC-1 cell lysates were included as controls. Total lysates from each cell line were resolved by 4–12% SDS-PAGE and immunoblotted with anti-ALCAM antibodies. Arrow indicates the fully glycosylated ALCAM isoform. Beta-actin was used as a loading control.</p

ALCAM expression in normal and tumor human thyroid tissue samples.

<p>(A) Total protein extracts (30 µg) from 11 papillary thyroid carcinomas (Ca Pap), 5 medullary thyroid carcinomas (Ca Mid), and 5 normal thyroid tissues (Thyroid) were resolved by 4–12% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with anti-ALCAM antibody. (*) indicates a non-specific background band. Beta-actin was used as a loading control. (B) Western blot analysis of ALCAM expression of total protein extracts (30 µg) from three PTCs, two MTCs, and three CTRLs, treated (+) or not (−) with N-glycosidase F. Beta-actin was used as a loading control.</p