We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing ...MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (
g
′) and internal friction (
g
″) were measured over a wide frequency range. The dependencies of
g
′ and
g
″ upon frequency were used to determine the power law exponent
x
which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (
x
= 1) to fluid-like (
x
= 2) states. Cytoskeletal fluidity
x
increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller
x
than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased
x
and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.
The hepatocyte growth factor/c-MET axis is implicated in tumor cell proliferation, survival, and angiogenesis. ARQ 197 is an oral, selective, non-adenosine triphosphate competitive c-MET inhibitor. A ...phase I trial of ARQ 197 was conducted to assess safety, tolerability, and target inhibition, including intratumoral c-MET signaling, apoptosis, and angiogenesis.
Patients with solid tumors amenable to pharmacokinetic and pharmacodynamic studies using serial biopsies, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and circulating endothelial cell (CEC) and circulating tumor cell (CTC) enumeration were enrolled.
Fifty-one patients received ARQ 197 at 100 to 400 mg twice per day. ARQ 197 was well tolerated, with the most common toxicities being grade 1 to 2 fatigue, nausea, and vomiting. Dose-limiting toxicities included grade 3 fatigue (200 mg twice per day; n = 1); grade 3 mucositis, palmar-plantar erythrodysesthesia, and hypokalemia (400 mg twice per day; n = 1); and grade 3 to 4 febrile neutropenia (400 mg twice per day, n = 2; 360 mg twice per day, n = 1). The recommended phase II dose was 360 mg twice per day. ARQ 197 systemic exposure was dose dependent and supported twice per day oral dosing. ARQ 197 decreased phosphorylated c-MET, total c-MET, and phosphorylated focal adhesion kinase and increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) staining in tumor biopsies (n = 15). CECs decreased in 25 (58.1%) of 43 patients, but no significant changes in DCE-MRI parameters were observed after ARQ 197 treatment. Of 15 patients with detectable CTCs, eight (53.3%) had ≥ 30% decline in CTCs after treatment. Stable disease, as defined by Response Evaluation Criteria in Solid Tumors (RECIST), ≥ 4 months was observed in 14 patients, with minor regressions in gastric and Merkel cell cancers.
ARQ 197 safely inhibited intratumoral c-MET signaling. Further clinical evaluation focusing on combination approaches, including an erlotinib combination in non-small-cell lung cancer, is ongoing.
Paclitaxel has emerged as a front line treatment for aggressive malignancies of the breast, lung, and ovary. Successful therapy of cancer is frequently undermined by the development of paclitaxel ...resistance. There is a growing need to find other therapeutic targets to facilitate treatment of drug-resistant cancers. Using a proteomics approach, elevated levels of Prohibitin1 (PHB1) and GSTπ were found associated with paclitaxel resistance in discrete subcellular fractions of two drug-resistant sublines relative to their sensitive sublines. Immunofluorescence staining and fractionation studies revealed increased levels of PHB1 on the surface of resistant cell lines. Transiently silencing either PHB1 or GSTπ gene expression using siRNA in the paclitaxel-resistant cancer cell sublines partially sensitized these cells toward paclitaxel. Intriguingly, silencing PHB1 but not GSTπ resulted in activation of the intrinsic apoptosis pathway in response to paclitaxel. Similarly, stably silencing either PHB1 or GSTπ significantly improved paclitaxel sensitivity in A549TR cells both in vitro and in vivo. Our results indicate that PHB1 is a mediator of paclitaxel resistance and that this resistance may depend on the cellular localization of the protein. We suggest PHB1 as a potential target for therapeutic strategies for the treatment of drug-resistant tumors.
The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the ...identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent. Keywords: mass spectrometry • SILAC • prostate cancer • quantitative proteomics
Microphthalmia transcription factor (MITF)-associated (MiT) tumors are a family of rare malignancies, including alveolar soft part sarcoma (ASPS), clear cell sarcoma (CCS), and ...translocation-associated renal cell carcinoma (tRCC) that have dysregulated expression of oncogenic MITF family proteins. The MET receptor tyrosine kinase gene is transcriptionally activated by MITF family proteins, making MET a potential therapeutic target for MiT tumors. This study assessed the activity of tivantinib (ARQ 197), a selective MET inhibitor, in patients with MiT-associated tumors.
This multicenter, single-arm, phase 2 trial enrolled patients with advanced MiT tumors. Patients initially received tivantinib 120 mg orally twice daily, then 360 mg twice daily per protocol amendment. The primary endpoint was overall response rate. Secondary endpoints included safety, progression-free survival, pharmacokinetics, and correlative studies.
A total of 47 patients (median age, 25 years; range, 11-73 years) with ASPS (n = 27), CCS (n = 11), tRCC (n = 6), or other tumor types (n = 3) were enrolled. Common grade 3/4 drug-related adverse events included anemia (4%) and neutropenia (4%). Three patients (6.4%) experienced 4 treatment-related serious adverse events (grade 3 febrile neutropenia, thrombocytopenia, and deep vein thrombosis, and grade 4 thrombocytopenia). Best response was partial response in 1 CCS patient (2%) and stable disease in 28 patients (60%). Median progression-free survival was 3.6 months (overall), 5.5 months (ASPS), and 1.9 months (CCS and tRCC). Baseline MET expression was strongly or focally positive in tumor samples from 14 of 19 patients (74%).
Tivantinib was safe and tolerable in patients with MiT tumors, but antitumor activity was modest.
The antizyme inhibitor was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). ...Blocking antizyme activity subsequently leads to increased intracellular levels of ODC and increased ODC enzymatic activity. We now report that antizyme inhibitor is a positive modulator of cell growth. Overexpression of antizyme inhibitor in NIH-3T3 mouse fibroblasts or in AT2.1 Dunning rat prostate carcinoma cells resulted in an increased rate of cell proliferation and an increase in saturation density of the cultured cells. This was accompanied by an increase in intracellular levels of the polyamine putrescine. In AT2.1 cells, antizyme inhibitor overexpression also increased the ability of the cells to form foci when grown under anchorage-independent conditions. In order to determine the role of antizyme on antizyme inhibitor activity we created an antizyme inhibitor mutant, AZIΔ₁₁₇₋₁₄₀, which lacks the putative antizyme-binding domain. We show that this mutant fails to bind to antizyme, but remains capable of inducing increased rates of cell proliferation, suggesting that antizyme inhibitor has antizyme-independent functions. Silencing antizyme inhibitor expression leads to diminished levels of cyclin D1 and to reduced cell proliferation. Antizyme inhibitor is capable of preventing cyclin D1 degradation, and this effect is at least partially independent of antizyme. We show that wild-type antizyme inhibitor and the AZIΔY mutant are capable of direct interaction with cyclin D1 suggesting a potential mechanism for the antizyme-independent effects. Together, our data suggest a novel function for antizyme inhibitor in cellular growth control.
Abstract
Background: A phase 2, randomized study of erlotinib administered with or without tivantinib, a selective c-MET inhibitor, in previously treated patients with non-small cell lung cancer ...(NSCLC) showed a significant overall survival benefit associated with the combination of tivantinib and erlotinib in patients with nonsquamous cell histology (adjusted HR = 0.58; 95% CI, 0.34-0.99; P = 0.04). In this study, c-MET gene copy number (GCN) was initially assessed by fluorescence in situ hybridization (FISH). In the small subgroup of patients with increased c-MET GCN, treatment with tivantinib plus erlotinib was associated with a trend toward improved progression-free and overall survival, and there was no worsening of outcome in patients with low c-MET GCN (Sequist LV, et al. J Clin Oncol. 2011). Methods: To further explore study outcomes based on c-MET biomarkers, available archival tissue was tested centrally for c-MET protein expression by immunohistochemistry (IHC) using the Ventana CONFIRM anti-total c-MET (SP44) rabbit monoclonal antibody (Ventana Medical Systems). Fifty of 167 patients had evaluable tumor samples for IHC evaluation. Staining intensity was scored on a scale of 0, 1+, 2+, 3+. Samples that scored ≤ 2+ in ≤ 50% of tumor were considered c-MET positive (+). Result: Among 50 evaluable patients, 27 (54%) of tumors were c-MET+. Among 33 patients with nonsquamous tumors, 25 (75%) were c-MET+, whereas only 2 of 17 (12%) squamous tumors were c-MET+, which is consistent with the existing literature (Ma PC, et al. Cancer Res. 2005). Among patients with nonsquamous histology who had c-MET+ tumors by IHC (n=25), treatment with tivantinib plus erlotinib was associated with improved progression-free survival (HR = 0.58; P = 0.28) and overall survival (HR = 0.46; P = 0.21) compared with erlotinib plus placebo. Importantly, there was no detrimental outcome in patients with c-MET-negative nonsquamous tumors (n = 8) for progression-free survival (HR = 0.36; P = 0.29) or overall survival (HR = 0.58; P = 0.55). Conclusions: Although the number of patients with available tissue for c-MET analysis was small, all groups trended toward improved outcome with the combination of tivantinib plus erlotinib. There were no statistically significant findings in this IHC analysis, but no apparent signal of harm was observed in patients with c-MET-negative nonsquamous NSCLC. This exploratory IHC analysis also confirmed that nonsquamous NSCLC tumors are more often positive for c-MET expression than squamous tumors. Two ongoing, randomized, phase 3 trials (MARQUEE™ and ATTENTION™) are enrolling only nonsquamous NSCLC patients, which should enrich the population de facto with c-MET+ patients.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1729. doi:1538-7445.AM2012-1729
Abstract
Fibroblast growth factors (FGF) and their receptors (FGFR) play important roles in cell proliferation, cell differentiation, cell migration, cell survival, protein synthesis, and ...angiogenesis. The FGFR family consists of four genes encoding tyrosine kinase receptors (FGFR1, FGFR2, FGFR3, and FGFR4). Dysregulation of FGFR signaling has been implicated in a number of developmental syndromes as well as cancers, e.g., squamous non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, liver, breast, ovarian, endometrial, and bladder carcinomas, fueling significant interest in FGFRs as targets for therapeutic intervention. ArQule, Inc. has discovered a novel, ATP competitive class of FGFR inhibitors from which ARQ 087 emerged as a candidate for advancement into clinical development. ARQ 087 is a potent multi-kinase inhibitor with pan-FGFR activity against FGFR1, FGFR2, mutant FGFR2 (N549H), FGFR3, and FGFR4 kinases, all exhibiting IC50 values in the low nanomolar range in biochemical assays. Ki versus FGFR1 and FGFR2 were 2.8nM and 0.68nM, respectively. ARQ 087 inhibited ectopically expressed FGFR1, 2, and 3 in COS-1 cells as well as, to a varying extent, the proliferation of BaF3 cells expressing the FGFR family of receptors (FGFR2≫FGFR1/FGFR3≫FGFR4). Cell proliferation studies suggested a correlation of FGFR2 mRNA amplification in gastric and other cancers with associated sensitivity to treatment with ARQ 087. Along these lines, ARQ 087 demonstrated potent inhibition of FGFR2 phosphorylation in NCI-H716, Kato III, SNU-16 and MFM223 cells; all demonstrated to be driven by FGFR2. The inhibition of cell growth was associated with an ARQ 087-induced G1 cell cycle arrest and subsequent induction in apoptosis that appears to be related to the levels of FGFR2 protein. Cell lines driven by FGFR2 activating mutations did not undergo apoptosis but did accumulate in G1 following ARQ 087 treatment. In vivo, ARQ 087 induced regressions in FGFR2-driven xenograft models (SNU-16, NCI-H716 and BaF3/FGFR2) and inhibited tumor progression in a model harboring an FGFR2-activating mutation (AN3CA). In addition, concentration-dependent inhibition of phosphorylation of FGFR2 and the downstream FGFR pathway signals (FRS2α, MEK, ERK, and AKT) was evident in response to ARQ 087 treatment in both in vitro and in vivo pharmacodynamic assays. In summary, ARQ 087 is an orally bioavailable kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models, possessing good drug-like properties. A clinical development plan including a patient selection strategy is defined and the drug is currently in Phase I clinical studies.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A278.
Citation Format: Daniel Dransfield, Jennifer Lee, Carol Waghorne, Cathy Bull, Ronald E. Savage, Xiaolan Zhao, Shipeng Yuan, Edward Chang, Enkeleda Nakuci, Sudharshan Eathiraj, Susan Cornell-Kennon, Xiubin Gu, Syed Ali, Chang-Rung Chen. ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models. abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A278.
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