{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T12:08:47Z","timestamp":1767182927432,"version":"build-2065373602"},"reference-count":49,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2014,5,16]],"date-time":"2014-05-16T00:00:00Z","timestamp":1400198400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAFV600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1].  In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAFV600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss,  with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1\/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.<\/jats:p>","DOI":"10.3390\/ijms15058773","type":"journal-article","created":{"date-parts":[[2014,5,16]],"date-time":"2014-05-16T12:09:04Z","timestamp":1400242144000},"page":"8773-8794","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K  Signaling Pathways"],"prefix":"10.3390","volume":"15","author":[{"given":"Inderjit","family":"Daphu","sequence":"first","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sindre","family":"Horn","sequence":"additional","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel","family":"Stieber","sequence":"additional","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, the Luxembourg Public Research Center for Health,  1445 Strassen, Luxembourg"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jobin","family":"Varughese","sequence":"additional","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Endy","family":"Spriet","sequence":"additional","affiliation":[{"name":"Molecular Imaging Center, Department of Biomedicine, University of Bergen, 5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hege","family":"Dale","sequence":"additional","affiliation":[{"name":"Molecular Imaging Center, Department of Biomedicine, University of Bergen, 5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kai","family":"Skaftnesmo","sequence":"additional","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rolf","family":"Bjerkvig","sequence":"additional","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"},{"name":"NorLux Neuro-Oncology Laboratory, the Luxembourg Public Research Center for Health,  1445 Strassen, Luxembourg"},{"name":"KG Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frits","family":"Thorsen","sequence":"additional","affiliation":[{"name":"NorLux Neuro-Oncology Laboratory, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"},{"name":"Molecular Imaging Center, Department of Biomedicine, University of Bergen, 5009 Bergen, Norway"},{"name":"KG Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen,  5009 Bergen, Norway"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2014,5,16]]},"reference":[{"key":"ref_1","first-page":"6997","article-title":"BRAF and RAS mutations in human lung cancer and melanoma","volume":"62","author":"Brose","year":"2002","journal-title":"Cancer Res"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1002\/cncr.21265","article-title":"CCI-779 in metastatic melanoma: A phase II trial of the California Cancer Consortium","volume":"104","author":"Margolin","year":"2005","journal-title":"Cancer"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1207","DOI":"10.1586\/ern.12.111","article-title":"Melanoma brain metastasis: Overview of current management and emerging targeted therapies","volume":"12","author":"Fonkem","year":"2012","journal-title":"Expert Rev. Neurother"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"812","DOI":"10.1016\/j.jconrel.2013.10.019","article-title":"Multimodal imaging enables early detection and characterization of changes in tumor permeability of brain metastases","volume":"172","author":"Thorsen","year":"2013","journal-title":"J. Control. Release"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.critrevonc.2004.08.006","article-title":"The pathogenesis and treatment of brain metastases: A comprehensive review","volume":"52","author":"Tosoni","year":"2004","journal-title":"Critical Rev. Oncol. Hematol"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e5857","DOI":"10.1371\/journal.pone.0005857","article-title":"The vascular basement membrane as \u201csoil\u201d in brain metastasis","volume":"4","author":"Carbonell","year":"2009","journal-title":"PLoS One"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Yajima, I., Kumasaka, M.Y., Thang, N.D., Goto, Y., Takeda, K., Yamanoshita, O., Iida, M., Ohgami, N., Tamura, H., and Kawamoto, Y. (2012). RAS\/RAF\/MEK\/ERK and PI3K\/PTEN\/AKT signaling in malignant melanoma progression and therapy. Dermatol. Res. Pract.","DOI":"10.1155\/2012\/354191"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1016\/j.bcp.2010.04.017","article-title":"Using genetics and genomics strategies to personalize therapy for cancer: Focus on melanoma","volume":"80","author":"Nathanson","year":"2010","journal-title":"Biochem. Pharmacol"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Pacheco, I., Buzea, C., and Tron, V. (2011). Towards new therapeutic approaches for malignant melanoma. Expert Rev. Mol. Med, 13.","DOI":"10.1017\/S146239941100202X"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1038\/nature00766","article-title":"Mutations of the BRAF gene in human cancer","volume":"417","author":"Davies","year":"2002","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1016\/j.bcp.2010.04.029","article-title":"Targeting the MAPK pathway in melanoma: Why some approaches succeed and other fail","volume":"80","author":"Inamdar","year":"2010","journal-title":"Biochem. Pharmacol"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2507","DOI":"10.1056\/NEJMoa1103782","article-title":"Improved survival with Vemurafenib in melanoma with BRAF V600E mutation","volume":"364","author":"Chapman","year":"2011","journal-title":"N. Engl. J. Med"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"7538","DOI":"10.1158\/1078-0432.CCR-09-1985","article-title":"Integrated molecular and clinical analysis of AKT activation in metastatic melanoma","volume":"15","author":"Davies","year":"2009","journal-title":"Clin. Cancer Res"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"67:1","DOI":"10.1186\/1479-5876-8-67","article-title":"Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032","volume":"8","author":"Rubinstein","year":"2010","journal-title":"J. Transl. Med"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1016\/j.hoc.2009.04.001","article-title":"BRAF signaling and targeted therapies in melanoma","volume":"23","author":"Dhomen","year":"2009","journal-title":"Hematol. Oncol. Clin. N. Am"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1658","DOI":"10.1158\/1078-0432.CCR-10-0174","article-title":"Targeting BRAF in Advanced Melanoma: A First Step toward Manageable Disease","volume":"17","author":"Vultur","year":"2011","journal-title":"Clin. Cancer Res"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2750","DOI":"10.1158\/0008-5472.CAN-10-2954","article-title":"PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression","volume":"71","author":"Paraiso","year":"2011","journal-title":"Cancer Res"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1046\/j.0022-202X.2004.22243.x","article-title":"Genetic interaction between NRAS and BRAF mutations and PTEN\/MMAC1 inactivation in melanoma","volume":"122","author":"Tsao","year":"2004","journal-title":"J. Invest. Dermatol"},{"key":"ref_19","first-page":"1800","article-title":"Relative reciprocity of NRAS and PTEN\/MMAC1 alterations in cutaneous melanoma cell lines","volume":"60","author":"Tsao","year":"2000","journal-title":"Cancer Res"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1016\/j.ccr.2010.11.023","article-title":"Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R\/PI3K","volume":"18","author":"Villanueva","year":"2010","journal-title":"Cancer Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1124\/jpet.112.201475","article-title":"Mechanisms limiting distribution of the threonine-protein kinase B-RaF(V600E) inhibitor dabrafenib to the brain: Implications for the treatment of melanoma brain metastases","volume":"344","author":"Mittapalli","year":"2013","journal-title":"J. Pharmacol. Exp. Ther"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1439","DOI":"10.1517\/13543784.2010.527945","article-title":"PLX4032: Does it keep its promise for metastatic melanoma treatment?","volume":"19","author":"Livingstone","year":"2010","journal-title":"Expert Opin. Investig. Drugs"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2029","DOI":"10.1038\/cdd.2012.94","article-title":"BH3-only protein silencing contributes to acquired resistance to PLX4720 in human melanoma","volume":"19","author":"Shao","year":"2012","journal-title":"Cell Death Differ"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2819","DOI":"10.2169\/internalmedicine.51.7675","article-title":"Long-term survival in metastatic malignant melanoma: Ipilimumab followed by vemurafenib in a patient with brain metastasis","volume":"51","author":"Balakan","year":"2012","journal-title":"Internal Med"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Dummer, R., Rinderknecht, J., Goldinger, S.M., Wagner, I., Mitchell, L., Veronese, M.L., Nick, S., Hilfiker, P., and Gobbi, S. (2011, January 3\u20137). An open-label pilot study of vemurafenib in previously treated metastatic melanoma patients with brain metastases. Chicago, IL, USA.","DOI":"10.1016\/S0923-7534(20)33679-6"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1001\/jamadermatol.2013.372","article-title":"Successful treatment with vemurafenib in BRAF V600K-positive cerebral melanoma metastasis","volume":"149","author":"Forschner","year":"2013","journal-title":"JAMA Dermatol"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Kolar, G.R., Miller-Thomas, M.M., Schmidt, R.E., Simpson, J.R., Rich, K.M., and Linette, G.P. (2013). Neoadjuvant treatment of a solitary melanoma brain metastasis with vemurafenib. J. Clin. Oncol, 31.","DOI":"10.1200\/JCO.2012.43.7061"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1016\/j.ejca.2013.11.002","article-title":"Vemurafenib in patients with BRAF (V600) mutation-positive melanoma with symptomatic brain metastases: Final results of an open-label pilot study","volume":"50","author":"Dummer","year":"2014","journal-title":"Eur. J. Cancer"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"7401","DOI":"10.1158\/1078-0432.CCR-07-0781","article-title":"Pharmacokinetic and Tumor Distribution Characteristics of Temsirolimus in Patients with Recurrent Malignant Glioma","volume":"13","author":"Kuhn","year":"2007","journal-title":"Clin. Cancer Res"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.1158\/1078-0432.CCR-11-2436","article-title":"Phase I Study of the Combination of Sorafenib and Temsirolimus in Patients with Metastatic Melanoma","volume":"18","author":"Davies","year":"2012","journal-title":"Clin. Cancer Res"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2445","DOI":"10.1158\/0008-5472.CAN-12-3514","article-title":"Automated tracking of nanoparticle-labeled melanoma cells improves the predictive power of a brain metastasis model","volume":"73","author":"Sundstrom","year":"2013","journal-title":"Cancer Res"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Hwang, S. (2012). Comparison and evaluation of pathway-level aggregation methods of gene expression data. BMC Genomics, 13.","DOI":"10.1186\/1471-2164-13-S7-S26"},{"key":"ref_33","unstructured":"Available online: http:\/\/www.ingenuity.com."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1038\/nature09454","article-title":"Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma","volume":"467","author":"Bollag","year":"2010","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"809","DOI":"10.1056\/NEJMoa1002011","article-title":"Inhibition of mutated, activated BRAF in metastatic melanoma","volume":"363","author":"Flaherty","year":"2010","journal-title":"N. Engl. J. Med"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3041","DOI":"10.1073\/pnas.0711741105","article-title":"Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity","volume":"105","author":"Tsai","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1517\/14728222.12.2.209","article-title":"Mammalian target of rapamycin as a therapeutic target in oncology","volume":"12","author":"Abraham","year":"2008","journal-title":"Expert Opin. Ther. Targets"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.drup.2007.11.003","article-title":"Targeting the PI3K\/Akt\/mTOR pathway: Effective combinations and clinical considerations","volume":"11","author":"LoPiccolo","year":"2008","journal-title":"Drug Resist. Updat"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1038\/modpathol.2009.109","article-title":"Characterization of candidate gene copy number alterations in the 11q13 region along with BRAF and NRAS mutations in human melanoma","volume":"22","author":"Ecsedi","year":"2009","journal-title":"Mod. Pathol"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5497","DOI":"10.1038\/onc.2008.245","article-title":"PI3K pathway alterations in cancer: Variations on a theme","volume":"27","author":"Yuan","year":"2008","journal-title":"Oncogene"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1406","DOI":"10.1093\/jnci\/81.18.1406","article-title":"Correlation of growth capacity of human tumor cells in hard agarose with their in vivo proliferative capacity at specific metastatic sites","volume":"81","author":"Li","year":"1989","journal-title":"J. Natl. Cancer Inst"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1606","DOI":"10.1200\/JCO.2006.06.0442","article-title":"Toward a molecular classification of melanoma","volume":"25","author":"Fecher","year":"2007","journal-title":"J. Clin. Oncol"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1001\/archderm.141.2.165","article-title":"Signaling networks in cutaneous melanoma metastasis identified by complementary DNA microarrays","volume":"141","author":"Nambiar","year":"2005","journal-title":"Arch. Dermatol"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1111\/j.1365-2990.2010.01119.x","article-title":"A novel brain metastases model developed in immunodeficient rats closely mimics the growth of metastatic brain tumours in patients","volume":"37","author":"Wang","year":"2011","journal-title":"Neuropathol. Appl. Neurobiol"},{"key":"ref_45","unstructured":"Available online: http:\/\/www.dsmz.de."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"5421","DOI":"10.1046\/j.1432-1327.2000.01606.x","article-title":"Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity","volume":"267","author":"Wilson","year":"2000","journal-title":"Eur. J. Biochem"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.ejphar.2010.12.004","article-title":"PTD4-apoptin protein and dacarbazine show a synergistic antitumor effect on B16-F1 melanoma in vitro and in vivo","volume":"654","author":"Jin","year":"2011","journal-title":"Eur. J. Pharmacol"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1177\/1087057106292763","article-title":"Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis","volume":"11","author":"Ivascu","year":"2006","journal-title":"J. Biomol. Screen"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1007\/s00401-013-1101-1","article-title":"EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis","volume":"125","author":"Talasila","year":"2013","journal-title":"Acta Neuropathol"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/15\/5\/8773\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:11:31Z","timestamp":1760217091000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/15\/5\/8773"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,5,16]]},"references-count":49,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2014,5]]}},"alternative-id":["ijms15058773"],"URL":"https:\/\/doi.org\/10.3390\/ijms15058773","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2014,5,16]]}}}