ABSTRACT
Title
Selumetinib sensitivity in human colorectal cancer and non-small cell lung cancer.
Authors
L.P. Ciuffreda1, S. Costantino1, T. Troiani2, R. Russo1, E. Martinelli2, F. Ciardiello2, F. Rossi1 and L. Berrino1.
1Department of Experimental Medicine, Pharmacology Division, Second University of Naples, Naples, Italy
2Department of Experimental Medicine, Oncology Division, Second University of Naples, Naples, Italy
1Department of Experimental Medicine, Pharmacology Division, Second University of Naples, Naples, Italy
2Department of Experimental Medicine, Oncology Division, Second University of Naples, Naples, Italy
Abstract
Propose:The RAS/RAF/MEK/ERK pathway is overactive in approximately 40% of colorectal cancer (CRC) and 20-30% of non-small cell lung cancer (NSCLC) and is thus a target for novel therapeutics. Selumetinib, a selective non-ATP-competitive inhibitor of MEK1/2 is under clinical development in several malignancies including CRC and NSCLC. The lack of the antitumor activity in clinical setting, despite what observed in preclinical models, highlights the importance of identifying patient population most likely to respond to this drug.
Aim:In this study, we evaluated the effects of selumetinib in vitro and in vivo in CRC and NSCLC to identify which characteristics correlate with sensitivity to MEK inhibition.
Methods: Five human non small lung cancer cell lines (GLC82; A549; Calu3; H460; H1299) and six human colon cancer cell lines (GEO; HCT15; HCT116; SW480; SW620; LS174T) were exposed to different concentration of selumetinib ( 0.01, 0.05, 0.25, 1, 5 and 10 µM) for 48, 72 and 96 hours. Cell proliferation was measured with the MTT assay. To confirm the sensitive and resistant phenotype of the cell lines in vivo, we used mouse xenograft model of 2-S (HCT116 and Calu3) and 2-R (HCT15 and H460) cell lines and treated them with two doses (25 mg/kg and 50 mg/kg BID) of selumetinib for 21 days. Up to 50 days following the treatment beginning the tumor volumw was measured (last day all control tumor volume were 2000 mm3). To identify different molecular patterns of sensitivity or resistance to Mek inhibition, RNAs from the 11 cell lines were extraxted and used for microarray experiments. The microarrays were scanned using Agilent G2565AA Microarray Scanner System and controlled by Agilent’s Scan Control Software version A 7.0.1. Chemiluminescent signals were quantified with Feature Extraction 9.5.3 software. Expression values were analyzed and ormalized by GeneSpring GX 11.5 using Student’s t test (p<0.05) with a Benjamani-Hochberg multiple test correction. Of the significantly differentially expressed RNA, only those with greater than 2-fold increase or decrease were considered. Functional and network analyses of statistically significant gene expression changes were performed using Ingenuity Pathways Analysis (IPA) 8.0 (Ingenuity® System, http: //www.ingenuity.com).
Results: We evaluated the sensitivity to the selective MEK1/2 inhibitor, selumetinib, in 5 NSCLC and 6 CRC cell lines using the MTT assay. The range of sensitivities was wide, with IC50s varied between 0.01 μM and >10 μM. We classified as sensitive (S) or resistant (R) cell lines showing IC50 at 96 hrs ≤ 1 μM or > 1 μM, respectively. In particular, 6 cell lines, 2 from NSCLC (Calu3 and H1299) and 4 from CRC (HCT116, GEO, SW480 and SW620), resulted sensitive.
As shown the mouse xenograft model the growth rate of the S cell lines was significantly decreased by selumetinib in a dose dependent manner. Moreover, selumetinib was unable to affect tumor growth in resistant cell lines , whose growth rate was similar to that of the control groups.
The gene expression experiment showed 21 up-regulated and 17 down-regulated genes in resistant cell lines. In particular, we found that among the up-regulated genes 6 were associated to PKA pathways.
Conclusion: These results indicate that resistance to selumetinib could be mediated, in part, by up regulation of the PKA pathway, suggesting rational combination partners for selumetinib.
Acknowledgements
This work was supported by Second University of Naples (Ricerca Ateneo).
Aim:In this study, we evaluated the effects of selumetinib in vitro and in vivo in CRC and NSCLC to identify which characteristics correlate with sensitivity to MEK inhibition.
Methods: Five human non small lung cancer cell lines (GLC82; A549; Calu3; H460; H1299) and six human colon cancer cell lines (GEO; HCT15; HCT116; SW480; SW620; LS174T) were exposed to different concentration of selumetinib ( 0.01, 0.05, 0.25, 1, 5 and 10 µM) for 48, 72 and 96 hours. Cell proliferation was measured with the MTT assay. To confirm the sensitive and resistant phenotype of the cell lines in vivo, we used mouse xenograft model of 2-S (HCT116 and Calu3) and 2-R (HCT15 and H460) cell lines and treated them with two doses (25 mg/kg and 50 mg/kg BID) of selumetinib for 21 days. Up to 50 days following the treatment beginning the tumor volumw was measured (last day all control tumor volume were 2000 mm3). To identify different molecular patterns of sensitivity or resistance to Mek inhibition, RNAs from the 11 cell lines were extraxted and used for microarray experiments. The microarrays were scanned using Agilent G2565AA Microarray Scanner System and controlled by Agilent’s Scan Control Software version A 7.0.1. Chemiluminescent signals were quantified with Feature Extraction 9.5.3 software. Expression values were analyzed and ormalized by GeneSpring GX 11.5 using Student’s t test (p<0.05) with a Benjamani-Hochberg multiple test correction. Of the significantly differentially expressed RNA, only those with greater than 2-fold increase or decrease were considered. Functional and network analyses of statistically significant gene expression changes were performed using Ingenuity Pathways Analysis (IPA) 8.0 (Ingenuity® System, http: //www.ingenuity.com).
Results: We evaluated the sensitivity to the selective MEK1/2 inhibitor, selumetinib, in 5 NSCLC and 6 CRC cell lines using the MTT assay. The range of sensitivities was wide, with IC50s varied between 0.01 μM and >10 μM. We classified as sensitive (S) or resistant (R) cell lines showing IC50 at 96 hrs ≤ 1 μM or > 1 μM, respectively. In particular, 6 cell lines, 2 from NSCLC (Calu3 and H1299) and 4 from CRC (HCT116, GEO, SW480 and SW620), resulted sensitive.
As shown the mouse xenograft model the growth rate of the S cell lines was significantly decreased by selumetinib in a dose dependent manner. Moreover, selumetinib was unable to affect tumor growth in resistant cell lines , whose growth rate was similar to that of the control groups.
The gene expression experiment showed 21 up-regulated and 17 down-regulated genes in resistant cell lines. In particular, we found that among the up-regulated genes 6 were associated to PKA pathways.
Conclusion: These results indicate that resistance to selumetinib could be mediated, in part, by up regulation of the PKA pathway, suggesting rational combination partners for selumetinib.
Acknowledgements
This work was supported by Second University of Naples (Ricerca Ateneo).