Intrinsic resistance to selumetinib, a selective inhibitor of MEK1/2, by cAMP-dependent protein kinase A activation in human lung and colorectal cancer cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: This study provides molecular insights to explain resistance to an MEK inhibitor in human cancer cell lines.

Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas.

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.

HMGI-C gene expression is not required for in vivo thyroid cell transformation.

We have previously demonstrated that HMGI proteins are required for the transformation of rat thyroid cells by v-mos and v-ras-Ki oncogenes. To determine whether HMGI proteins are also required for in vivo thyroid carcinogenesis, mice carrying a disrupted HMGI-C gene (pygmy mice) were either treated with radioactive iodine or crossed with transgenic mice carrying the E7 papilloma virus oncogene under the transcriptional control of thyroglobulin gene promoter. The pygmy mice developed thyroid carcinomas with the same frequency as occurred in wild-type mice without significant macroscopic and microscopic differences. Therefore, these results indicate that HMGI-C gene expression is not required in in vivo thyroid cell malignant transformation.

Human N-ras, TRK-T1, and RET/PTC3 oncogenes, driven by a thyroglobulin promoter, differently affect the expression of differentiation markers and the proliferation of thyroid epithelial cells.

The ras family members and the tyrosine kinases RET and TRK are frequently activated in human tumors of the thyroid gland. To ascertain the effects of these oncogenes in cultured thyroid cells we have generated expression vectors containing activated versions of the three genes under the control of the thyroid-specific thyroglobulin gene promoter. Here we show that the expression of the three oncogenes differently affects thyroid differentiation. While the TRK-T1 oncogene interferes with the capability of thyroid cells of trapping iodide and only marginally affects thyroglobulin gene expression, both RET/PTC3 and N-ras(Gln61-Lys) induce a dramatic reduction of thyroglobulin mRNA and alleviate TSH dependency for cellular growth. However, none of the three oncogenes is able to induce the appearance of neoplastic transformation markers, such as growth in semisolid medium and tumorigenicity in athymic mice. This indicates that genetic events additional to TRK, RET, or N-ras activation are required for fully malignant transformation of thyroid cells.

TPA induces a block of differentiation and increases the susceptibility to neoplastic transformation of a rat thyroid epithelial cell line.

The PC Cl 3 cell line is a well-characterized epithelial cell line of rat thyroid origin. This cell line retains in vitro the typical markers of thyroid differentiation: thyroglobulin (TG) synthesis and secretion, iodide uptake, thyroperoxidase (TPO) expression, and dependency on TSH for growth. Although the differentiated phenotype of thyroid cells has been relatively well described, the molecular mechanisms that regulate both differentiation and neoplastic transformation of thyroid cells still need to be investigated in detail. Protein kinase C (PKC), the target of tetradecanoylphorbol acetate (TPA), regulates growth and differentiation of several cell types. Here we show that treatment of PC Cl 3 cells with TPA induces an acute block of thyroid differentiation. TPA-treated PC Cl 3 cells are unable to trap iodide and the expression levels of thyroglobulin, TSH receptor, and TPO genes are drastically reduced by TPA treatment. This differentiation block is not caused by a reduced expression of one of the master genes of thyroid differentiation, the thyroid transcription factor 1 (TTF-1). TPA-treated PC Cl 3 cells display an increased growth rate indicating that, in addition to the differentiation block, TPA also significantly affects the growth regulation of thyroid cells. Finally, TPA treatment dramatically increases the number of transformation foci induced in PC Cl 3 cells by retroviruses carrying v-Ki-ras, v-Ha-ras, and v-mos oncogenes. These findings support the notion that the PKC pathway can influence proliferation, differentiation, and neoplastic transformation of thyroid cells in culture.

Benefits of monitoring plasma levodopa in Parkinson's disease patients with drug-induced chorea.

We studied the temporal patterns of chorea and plasma levodopa profiles in 30 patients with Parkinson's disease whose motor fluctuations were difficult to characterize and treat on the basis of observation alone. We were able to determine whether chorea was associated with high levodopa concentrations or low levodopa concentrations or both. We found the following patterns of levodopa-associated chorea: chorea due to inadequate levodopa levels, chorea due to biphasic levodopa absorption, chorea associated with either rapid or slow levodopa absorption, and chorea due to long-duration levodopa absorption mimicking a sustained-release preparation. Seven patients benefited after their dosing schedules were rearranged as a result of information gained from monitoring. We conclude that any patient with levodopa-associated chorea who cannot be regulated on the basis of observation alone should be studied with simultaneous plasma levodopa measurements and clinical monitoring to detect an unusual plasma levodopa pattern that may be improved by adjustment of dosing schedule.

Complex dystonia of Parkinson's disease: clinical features and relation to plasma levodopa profile.

We studied the clinical characteristics and plasma levodopa (LD) profile of complex, LD-associated dystonia in 33 patients with Parkinson's disease (PD). Generalized, abdominal, respiratory, and myoclonic, but not facial or foot, dystonia could be related to specific phases of the plasma LD cycle. Simultaneous clinical observation and determinations of plasma LD concentrations were often necessary to find the most efficacious dosing schedule for these patients. Maintenance of steady plasma LD concentrations within a dystonia-free window of benefit was the best treatment.

Continuous levodopa infusions to treat complex dystonia in Parkinson's disease.

We report the clinical spectrum of 3 patients with Parkinson's disease who experienced complex patterns of levodopa-related dystonia. Dystonia was unrelieved by multiple medication regimens but responded well to continuous, duodenal levodopa infusions. Patients were able to remain mobile without severe dystonia despite a very narrow window of benefit between the levodopa concentration necessary to achieve the "on" state and that which caused the onset of dystonic spasms.