Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Oncotarget
2015 Nov 24;637:40255-67. doi: 10.18632/oncotarget.5434.
Show Gene links
Show Anatomy links
Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells.
Oh SJ
,
Noh KH
,
Lee YH
,
Hong SO
,
Song KH
,
Lee HJ
,
Kim S
,
Kim TM
,
Jeon JH
,
Seo JH
,
Kim DW
,
Kim TW
.
Abstract
The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.
Figure 1. EML4-ALK increases the stem-like properties and tumorigenicity of EML4-ALK-driven NSCLC cells in vitro and in vivoA. Sphere-forming capacity of BEAS-2B, A549, H3122 and H2228 cells in a low-density suspension culture. Original magnification, x40. B. ALK, pALK, NANOG, OCT4, SOX2, KLF4, c-MYC and β-ACTIN expression in BEAS-2B, A549, H3122 and H2228 cells in a low-density suspension culture. Original magnification, x40. (B) ALK, pALK, NANOG, OCT4, SOX2, KLF4, c-MYC and β-ACTIN expression in BEAS-2B, A549, H3122 and H2228 cells was visualized by western blot analysis with lysates from the monolayer cultured cells. (C) H3122 and H2228 cells were treated with siGFP (control) or siALK and the levels of ALK, NANOG, OCT4, SOX2, KLF4, and c-MYC proteins were analyzed. β-ACTIN was used as an internal loading control. Numbers below blots indicate expression as measured by fold change. D. Flow cytometry analysis of the frequency of ALDH1+ cells in H3122 and H2228 cells treated with siALK or siGFP (control). E. Sphere-forming capacity of H3122 and H2228 cells treated with siGFP or siALK in a low-density suspension culture. Original magnification, × 40. F. Tumorigenicity of siGFP-versus siALK-treated H3122 cells inoculated at indicated doses into 5 NOD/SCID mice per group. G. Tumors were extracted at 20 days after injection of 105
siGFP- or siALK-treated H3122 cells. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t-test.
Figure 2. Ectopic expression of EML4-ALK enhances the stem-like properties and tumorigenicity of EML4-ALK negative cellsA. A549 cells were transfected with an empty vector or EML4-ALK variant 1 (EAV1) cDNA and the levels of ALK, pALK, NANOG, and OCT4 were analyzed. β-ACTIN was used as an internal loading control. Numbers below blots indicate expression as measured by fold change. B. Flow cytometry analysis of the frequency of ALDH1+ cells in A549 cells transfected with EAV1 or empty vector. C. Sphere-forming capacity of EAV1 versus empty vector-transfected A549 cells in a low-density suspension culture. Original magnification, × 40. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t-test.
Figure 3. Crizotinib, an ALK inhibitor, reduces the stem-like properties of EML4-ALK positive cells in a dose-dependent mannerH3122 cells were treated with crizotinib at the indicated concentration. A. ALK, pALK, NANOG, OCT4, and β-ACTIN expression in H3122 cells treated with crizotinib or DMSO (control) for 24 hr was visualized by western blot analysis. Numbers below blots indicate expression as measured by fold change. B. Flow cytometry analysis of the frequency of ALDH+ cells in H3122 cells treated with crizotinib or DMSO (control) for 24 hr. C. Sphere-forming capacity of H3122 cells treated with crizotinib or DMSO (control) in a low-density suspension culture. Original magnification, × 40. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t-test.
Figure 4. Rapamycin is the most suitable drug that decreases the stem-like properties of EML4-ALK-driven NSCLC cellsH3122 cells were treated with rapamycin, salinomycin, and metformin at the indicated concentrations. A. H3122 cells were treated with DMSO, rapamycin, salinomycin, or metformin for 24 hr, and the levels of NANOG, OCT4, and β-ACTIN protein were analyzed. Numbers below blots indicate expression as measured by fold change. B. Flow cytometry analysis of the frequency of ALDH+ cells in H3122 cells treated as in (A). C. Quantification of tumorsphere formation with H3122 cells treated with the indicated drugs in a low-density suspension culture. Original magnification, × 40. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t-test.
Figure 5. Inhibition of EML4-ALK-mediated stem-like properties enhances the anti-tumor effectA. Western blot analysis using antibodies specific to the proteins in lysates from H3122 cells that treated with the indicated drugs for 24 hr. Numbers below blots indicate expression as measured by fold change. B. Flow cytometry analysis of the frequency of ALDH+ cells in H3122 cells treated as in (A). C. Quantification of tumorsphere-formation with H3122 cells treated with the indicated drugs in a low-density suspension culture. Original magnification, × 40. D. Tumor growth in mice inoculated with H3122 cells. Nude mice were inoculated subcutaneously with 1 × 106 cells/mouse. Nine days following tumor challenge, the CH containing the indicated drug or both drugs was injected intratumorally. E. Tumor weight in mice at 23 days after the challenge. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t-test.
Figure 6. Rapamycin can effectively reduce the stem-like properties of crizotinib-resistant cellsA. Western blot analysis using antibodies specific to the proteins in lysates from H3122 cells or H3122 CR1 cells. Numbers below blots indicate expression as measured by fold change. B. and C. Dose-response curves for the viability of control H3122 cells and H3122 CR1 cells treated with crizotinib or rapamycin for 72 hr. D. Western blot analysis using antibodies specific to the proteins in lysates from H3122 CR1 cells that treated with the indicated drugs for 24 hr. Numbers below blots indicate expression as measured by fold change. E. Flow cytometry analysis of the frequency of ALDH+ cells in H3122 CR1 cells treated as in (D). F. Quantification of tumorsphere-formation with H3122 CR1 cells treated with the indicated drugs in a low-density suspension culture. Original magnification, × 40. G. Tumor growth in mice inoculated with H3122 CR1 cells. Nude mice were inoculated subcutaneously with 1 × 106 cells/mouse. Nine days following tumor challenge, the CH containing the indicated drug or both drugs was injected intratumorally. H. Tumor weight in mice at 23 days after challenge. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t-test.
Ben-Porath,
An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors.
2008, Pubmed
Ben-Porath,
An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors.
2008,
Pubmed
Bonnet,
Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.
1997,
Pubmed
Chiou,
Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma.
2008,
Pubmed
Choi,
EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors.
2010,
Pubmed
,
Echinobase
Christensen,
Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma.
2007,
Pubmed
Dalerba,
Cancer stem cells: models and concepts.
2007,
Pubmed
Doebele,
Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer.
2012,
Pubmed
Du,
Pancreatic cancer cells resistant to chemoradiotherapy rich in "stem-cell-like" tumor cells.
2011,
Pubmed
Dylla,
Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy.
2008,
Pubmed
Ginestier,
ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.
2007,
Pubmed
Guertin,
Defining the role of mTOR in cancer.
2007,
Pubmed
Gupta,
Identification of selective inhibitors of cancer stem cells by high-throughput screening.
2009,
Pubmed
Hallberg,
Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology.
2013,
Pubmed
Hay,
Upstream and downstream of mTOR.
2004,
Pubmed
Hirsch,
Metformin inhibits the inflammatory response associated with cellular transformation and cancer stem cell growth.
2013,
Pubmed
Horn,
EML4-ALK: honing in on a new target in non-small-cell lung cancer.
2009,
Pubmed
Iliopoulos,
Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types.
2011,
Pubmed
Janku,
Targeted therapy in non-small-cell lung cancer--is it becoming a reality?
2010,
Pubmed
Katayama,
Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers.
2012,
Pubmed
Kim,
Heterogeneity of genetic changes associated with acquired crizotinib resistance in ALK-rearranged lung cancer.
2013,
Pubmed
Kreso,
Evolution of the cancer stem cell model.
2014,
Pubmed
Kwak,
Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
2010,
Pubmed
Lee,
CD24(+) liver tumor-initiating cells drive self-renewal and tumor initiation through STAT3-mediated NANOG regulation.
2011,
Pubmed
Lee,
Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway.
2010,
Pubmed
Li,
Clinical significance of EML4-ALK fusion gene and association with EGFR and KRAS gene mutations in 208 Chinese patients with non-small cell lung cancer.
2013,
Pubmed
Li,
RIG-I modulates Src-mediated AKT activation to restrain leukemic stemness.
2014,
Pubmed
Ma,
CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway.
2008,
Pubmed
Martelli,
Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.
2011,
Pubmed
Matsubara,
mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions.
2013,
Pubmed
Morán,
Targeting EML4-ALK driven non-small cell lung cancer (NSCLC).
2013,
Pubmed
Mueller,
Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer.
2009,
Pubmed
Noh,
Nanog signaling in cancer promotes stem-like phenotype and immune evasion.
2012,
Pubmed
Parker,
Enhanced epidermal growth factor, hepatocyte growth factor, and vascular endothelial growth factor expression in tuberous sclerosis complex.
2011,
Pubmed
Reya,
Stem cells, cancer, and cancer stem cells.
2001,
Pubmed
Rosell,
Lung cancer: Maintenance therapy and precision medicine in NSCLC.
2013,
Pubmed
Sabatini,
mTOR and cancer: insights into a complex relationship.
2006,
Pubmed
Sarbassov,
Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.
2005,
Pubmed
Sasaki,
A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors.
2011,
Pubmed
Sasaki,
The biology and treatment of EML4-ALK non-small cell lung cancer.
2010,
Pubmed
,
Echinobase
Soda,
Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.
2007,
Pubmed
,
Echinobase
Soda,
A mouse model for EML4-ALK-positive lung cancer.
2008,
Pubmed
Sullivan,
Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling.
2010,
Pubmed
Tu,
Targeting stem cells-clinical implications for cancer therapy.
2009,
Pubmed
Visvader,
Cancer stem cells in solid tumours: accumulating evidence and unresolved questions.
2008,
Pubmed
Zhou,
Tumour-initiating cells: challenges and opportunities for anticancer drug discovery.
2009,
Pubmed