Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor ...receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.
MEK is activated in ∼40% colorectal cancer (CRC) and 20-30% non-small cell lung cancer (NSCLC). Selumetinib is a selective inhibitor of MEK1/2, which is currently in clinical development.
We ...evaluated the effects of selumetinib in vitro and in vivo in CRC and NSCLC cell lines to identify cancer cell characteristics correlating with sensitivity to MEK inhibition.
Five NSCLC and six CRC cell lines were treated with selumetinib and classified according to the median inhibitory concentration (IC(50)) values as sensitive (≤1 μM) or resistant (>1 μM). In selumetinib-sensitive cancer cell lines, selumetinib treatment induced G1 cell-cycle arrest and apoptosis and suppression of tumour growth as xenografts in immunodeficient mice. Evaluation of intracellular effector proteins and analysis of gene mutations showed no correlation with selumetinib sensitivity. Microarray gene expression profiles revealed that the activation of cAMP-dependent protein kinase A (PKA) was associated with MEK inhibitor resistance. Combined targeting of both MEK and PKA resulted in cancer cell growth inhibition of MEK inhibitor-resistant cancer cell lines in vitro and in vivo.
This study provides molecular insights to explain resistance to an MEK inhibitor in human cancer cell lines.
Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene ...(Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.
Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by ...onset of resistance.
We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab.
Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET-dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR-MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells.
These results suggest that overexpression of TGF-α through induction of EGFR-MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer.
The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these ...pathways have been developed as molecular targeted anti‐cancer therapies. The in vitro and in vivo anti‐tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi‐targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib‐sensitive (IC50 for cell growth inhibition of 0.001 µM) or pimasertib‐resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up‐regulated in pimasertib‐resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK‐ and AKT‐dependent signaling pathways in pimasertib‐resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.
What's new?
Drugs that block key regulators of cell proliferation and survival form the basis of targeted anticancer therapy but are highly susceptible to resistance‐conferring mutations in their targets. This limitation may be overcome with various combinations of drugs, as shown here with pimasertib, a small‐molecule MEK1/MEK2 inhibitor. Microarray analysis reveals that the combination of pimasertib with PI3K/mTOR or multi‐targeted kinase inhibitors prevents proliferation and induces apoptosis in pimasertib‐resistant HCT15 colorectal cancer and H1975 non‐small cell lung cancer cells. Combined blockade of key intracellular pathways produced similar effects in mice with tumors established from the two pimasertib‐resistant lines.
Purpose: Vascular endothelial growth factor (VEGF) is a major mitogen for endothelial cells and enhances vascular permeability. Enhanced
VEGF secretion is found in human cancers and correlates with ...increased tumor neovascularization. ZD6474 is a p.o. bioavailable,
VEGF flk-1/KDR receptor (VEGFR-2) tyrosine kinase inhibitor with antitumor activity in many human cancer xenografts and is
currently in Phase I clinical development.
Experimental Design: We tested the effects of ZD6474 on EGFR phosphorylation in cell expressing functional epidermal growth factor receptor (EGFR)
and the antiproliferative and the proapoptotic activity of ZD6474 alone or in combination taxanes in human cancer cell lines
with functional EGFR but lacking VEGFR-2. The antitumor activity of this drug was also tested in nude mice bearing established
GEO colon cancer xenografts.
Results: ZD6474 causes a dose-dependent inhibition of EGFR phosphorylation in mouse NIH-EGFR fibroblasts and human MCF-10A ras breast
cancer cells, two cell lines that overexpress the human EGFR. ZD6474 treatment resulted in a dose-dependent inhibition of
soft agar growth in seven human cell lines (breast, colon, gastric, and ovarian) with functional EGFR but lacking VEGFR-2.
A dose-dependent supra-additive effect in growth inhibition and in apoptosis in vitro was observed by the combined treatment with ZD6474 and paclitaxel or docetaxel. ZD6474 treatment of nude mice bearing palpable
GEO colon cancer xenografts (which are sensitive to inhibition of EGFR signaling) induced dose-dependent tumor growth inhibition.
Immunohistochemical analysis revealed a significant dose-dependent reduction of neoangiogenesis. The antitumor activity of
ZD6474 in GEO tumor xenografts was also found to be enhanced when combined with paclitaxel. Tumor regression was observed
in all mice after treatment with ZD6474 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition
of angiogenesis. Six of 20 mice had no histological evidence of tumors after treatment with ZD6474 plus paclitaxel.
Conclusions: This study suggests that in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling, ZD6474
may also be able to inhibit cancer cell growth by blocking EGFR autocrine signaling. These results provide also a rationale
for the clinical evaluation of ZD6474 combined with taxanes in cancer patients.
RET/papillary thyroid carcinoma (PTC) oncogenes, generated by recombination of the tyrosine kinase-encoding domain of RET with different heterologous genes, are prevalent in papillary carcinomas of ...the thyroid. Point mutations of RET cause multiple endocrine neoplasia type 2 (MEN2) familial cancer syndrome and are found in sporadic medullary thyroid carcinomas. Here, we show that ZD6474, a low molecular weight tyrosine kinase inhibitor, blocks the enzymatic activity of RET-derived oncoproteins at a one-half maximal inhibitory concentration of 100 nM. ZD6474 blocked in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins and of an epidermal growth factor (EGF)-activated EGF-receptor/RET chimeric receptor. RET/PTC3-transformed cells-treated ZD6474 lost proliferative autonomy and showed morphological reversion. ZD6474 prevented the growth of two human PTC cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, it blocked anchorage-independent growth of RET/PTC3-transformed NIH3T3 fibroblasts and the formation of tumors after injection of NIH-RET/PTC3 cells into nude mice. Thus, targeting RET oncogenes with ZD6474 might offer a potential treatment strategy for carcinomas sustaining oncogenic activation of RET.
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