Introduction
Lung cancer is the leading cause of cancer deaths in the world. More patients die from lung cancer than breast, colon, prostate, and kidney cancer combined. Approximately 85% of patients diagnosed with lung cancer will die from their disease. Lung cancers initially responding to chemotherapeutic agents will eventually develop resistance to therapy. The expression of stem markers Oct4 and/or nestin in cancer cells is associated with resistance to chemotherapeutic agents leading to treatment failures [
1‐
5].
Cancer stem cells (CSC) have been defined as rare tumor cells with the capacity to self-renewal and initiate tumor growth in mouse xenografts that histologically recapitulate the primary tumor [
6,
7]. CSC are reported to be more resistant to chemotherapy agents and the induction of apoptosis compared to other populations of cells within the same tumor [
8‐
11]. Self-renewal and chemotherapy resistance in cancer-initiating cells is mediate through the expression of inhibitor of differentiation/DNA binding proteins Id1 and Id3 [
12‐
14].
CD44 and CD133 antigens are commonly used to isolate CSC from lung and other carcinomas [
7,
11,
15‐
19]. Isolated CD44 and CD133 cancer cells also express stem cell regulators Oct4, Sox2, nanog, and nestin [
11,
20‐
23]. Oct4 is transiently expressed during early development in pluripotent stem cells and is required for self-renewal [
24]. Nestin is a marker of neural progenitor cells and is frequently expressed in cancer cells of non-small cell lung carcinomas [
21,
25‐
27]. Although several studies have shown CD44 + and CD133 + cells initiate tumor growth at a significantly lower number of cells compared to the negative populations, CD44- and CD133- populations have also been reported be tumor initiating cells in some studies [
17,
28]. These studies suggest that further characterization of specific population of cancer cells may be needed.
Self-renewal is an essential mechanism required for stem cells to maintain long-term populating cells. Bone morphogenetic proteins 2 and 4 (BMP2/4) mediate self-renewal of embryonic stems by stimulating the expression of Id1 [
29]. BMPs signal through transmembrane serine/kinases composed of type I (alk2, alk3, and alk6) and type II receptors. The BMP receptor complex phosphorylates smad-1/5, which then activates response elements on the Id1, Id2, and Id3 promoters [
30,
31]. Downregulation of type I BMP receptors with siRNA and selective small molecule antagonists decreases the phosphorylation of smad-1/5 causing a decrease in expression of Id, Id2, and Id3 in lung cancer cell lines [
32]. The inhibition of BMP type I receptors also induces cell death and causes significant growth inhibition of lung cancer cell lines, which is mediated through the downregulation of Id proteins [
32]. The role of the BMP signaling cascade regulating the expression of Id proteins and growth of cancer cells expressing Oct4 or nestin is not known.
We further delineate the heterogeneity of lung cancer by showing that Oct4, nestin, and Neun are expressed in lung cancer cell lines and primary lung tumors. We isolated from lung cancer cell lines, cells that express Oct4 or nestin. Our studies suggest that Oct4 and nestin expressing cancer cells are a different population of tumor-initiating cells. Inhibition of BMP signaling with the selective antagonist DMH2 caused a decrease in the expression of Id1/Id3 and induced significant growth inhibition of cancer cells expressing Oct4 or nestin. Blockade of BMP signaling with small molecule antagonists of the type I BMP receptors represents a potential means to regulate the growth of lung cancer cells expressing stem cell markers.
Materials and methods
Cell culture
The A549 and H1229 lung cancer cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Sigma Aldrich, St Louis, MO, USA) with 5% fetal bovine serum (FBS) [
33]. The lung cancer cell lines H157, H727, U1752, and H358, and H865 were cultured in 90% RPMI and 10% FCS. The cell lines were obtained from ATCC and from Malcolm Brock, John Hopkins University.
Expression vectors
The Oct4 promoter/EGFP plasmid vector was a gift from Wei Cui (Roslin Institute, Midiothian, UK [
34]. The nestin promoter/EGFP was obtained from Rohan Humphrey (La Jolla, CA). The SM22 promoter/luciferase expression vector was obtained from Julian Solway (University of Chicago, Chicago IL) [
35]. The SM22 promoter was cloned into the pAcGFP 1–1 expression vector at the XhoI/Hind III sites (Clontech, Palo Alto, CA). Cells were transfected using electroporation and then selected with neomycin. Control cells were transfected with pcDNA 3.1 vector (Invitrogen) expressing EGFP (Clontech).
Human tumor samples
Human lung tumor tissue samples were obtained from the Rutgers Cancer Institute of New Jersey (CINJ) after approval by the institutional review board and ethics committee of the Rutgers Robert Wood Johnson Medical School. Protocol approval number, 0220013730. The review committee waived the need for consent since no patient identifiers were used.
Cell death assay
Cells were plated in 6 well plates at 106 cells per well and treated with 1 μM DMSO or 1 μM DMH2 for 48 hours. Adherent and floating cells were harvested and incubated with 0.1 mg/ml of ethidium bromide. Immediately after staining approximately 100 cells were counted and the percentage of cells that took up ethidium bromide was determined.
Cell counts
Cells were plated into 6 well plates at 105 cells per well and treated with 1 μM DMSO or 1 μM DMH2 for 7 days. The cells were detached with trypsin, stained with trypan blue, and the number of live cells counted using a hemacytometer.
Immunoflourescent imaging
Immunofluorescent imaging was performed on both non-adherent and adherent cells as previously described [
36]. Cells were trypsinized and immunofluorescent imaging performed or placed into cloning chambers (Nunc Lab-Tek, Rochester, NY). Briefly, cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X, and blocked with 1% BSA/PBS. Cells were incubated with primary antibodies in 1X PBS/1% BSA at room temperature for one hour. Appropriate Alex Fluor 488, 568, or 647 (Invitrogen/Molecular Probes) conjugated secondary antibodies were used. The secondary antibody was added for one hour at room temperature. Controls were treated in the same manner but did not receive primary antibody. In all negative controls samples there was no fluorescent signal. Primary antibodies used were rabbit anti-Oct 4 (Santa Cruz, Santa Cruz, CA), rabbit anti-human nestin (Chemicon), mouse anti-human nestin (Chemicon), and mouse anti-NeuN (Chemicon, Temecula, CA). Fluorescent images were captured using a Nikon Eclipse TE 300 inverted epifluorescent microscope and a Cool Snap black and white digital camera. IP Lab imaging software was used to assign pseudo-color to each channel.
Immunohistochemistry (IHC)
IHC was performed on formalin-fixed paraffin-embedded primary NSCLC and tumor xenografts in mice. Antibodies used were mouse anti-Oct4A (Cell Marque, Rocklin, CA), mouse anti-human nestin (Chemicon), mouse anti-NeuN (Chemicon, Temecula, CA), and mouse anti-smooth muscle actin (SMA) (clone 1A4) (Sigma, St. Louis, MI). IHC was performed on 5 μm tissue sections. Detection of Oct4 and NeuN on primary NSCLC used Tris-EDTA antigen retrieval using Vantana Benchmark XT automated IHC system. Seminoma was used as a positive control for Oct4 and normal brain for NeuN. For detection of nestin, NeuN, and SMA antigen retrieval was performed using Target Retrieval Solution (Dako Cytomation, Carpentaria, CA). On these samples, the Biomodule IHC Staining Kit (Invitrogen) was used as per the manufacture’s instructions. IHC on cell lines was performed by plating cells on glass cover slips, fixing in 4% paraformaldehyde for 10 minutes, incubating with primary Oct4 antibody for 1 hour, and using the biomodule IHC staining kit for detection.
Quantification of gene expression
RNA was extracted using the RNeasy kit as per the manufacturer's instructions (Qiagen, Valencia, CA). DNAase was used to remove any DNA contamination. cDNA was generated using Advantage RT for PCR kit (BD BiosciencesClontech, Palo Alto, CA). Quantitative PCR was performed with the Stratagene Mx3005p real-time thermal cycler (Agilent Technologies) with predesigned and validated Taq-Man gene expression assays according to the manufacturer’s specifications (Life Technologies, Grand Island, NY). Reference numbers used are: GAPDH (Hs99999905_m1), actin (99999903_m1), ACVRL1 (alk1) (Hs00163543_m1), ACVR1A (alk2) (Hs00153836_m1), BMR1A (alk3) (Hs00831730_s1), BMPR1B (alk6) (Hs00176144_m1), Pou3f1 (Hs00538614_s1) CD133 (Hs01009250_m1), UBE2Q1 (Hs01079904_m1), Pank3 (Hs00388176_g1), and Sel1L (Hs01071406_m1), Negative control included all reagents except cDNA. Expression was normalized to GAPDH using the formula 2∆ CT.
SYBER Green was used to detect double-stranded DNA for the following primers. Nestin (F) 5′-GCC-CTG-ACC-ACT-CCA-GTT-TA-3′ (R) 5′-GGA-GTC-CTG-GAT-TTC-CTT-CC-3′, Sox-2 (F) 5′-CAT-CAC-CCA-CAG-CAA-ATG-AC-3′ (R) 5′-TGC-AAA-GCT-CCT-ACC-GTA-CC-3′. Oct4A specific primers were (F) 5′-TCC-CTT-CGC-AAG-CCC-TCA-T-3′ and (R) 5′-TGA-CGG-TGC-AGG-GCT-CCG-GGG-AGG-CCC-CAT-C-3′. Oct4 primers spanning the first intron were (set 2) (F) 5′-GAA-GCT-GGA-GAA-GGA-GAA-GC- 3′. (R) 5′-GCC-GGT-TAC-AGA-ACC-ACA-CT-3′. PCR products were run on a gel, cDNA purified, and sequenced (GENEWIZ, South Plainfield, NJ). Genomic contamination was examined by quantitative PCR of RNA samples. Negative control included all reagents except cDNA.
Transient gene knockdown
Silencer Select Validated siRNA was used to knockdown expression of Oct4 (Life Technologies, Grand Island,NY), ID number S10871. Silencer Select Negative Control siRNA (4390843) was used to confirm specificity of knockdown. One million H1299 cells were transfected with 30 nM siRNA with the Nucleofector II (Amaxa Biosystems, Gaitherburg, MD) using the manufacture’s Nucleofector kit T. Optimization was performed using the enhanced green fluorescent reporter (EGFP) (Clontech) expressed in the pcDNA 3.1 vector (Invitrogen), which showed approximately 80% of the cells were transfected using this transfection protocol. Fourty-eight hours after transfection the expression of Oct4 expression was examined by quantitative PCR and Western blot analysis.
Microarray
By FACS, 106 cells expressing high levels of GFP were isolated from H1299 cells stably expressing the Oct4 promoter/GFP or Nestin promoter /GFP reporter vectors. After 24 hours total RNA was isolated using RNeasy Mini Kit as described by the manufacturer (Qiagen). DNAse treated RNA concentration was measured using NanoDrop 1000 spectrophotometer (Thermo Scientific) and the quality was analyzed with Bioanalyzer 2100 (Agilent). Spotted microarrays were used to identify differentially expressed genes between the Oct4/GFP and Nestin/GFP cells. After reverse transcription with SuperScript II, cDNA was transcribed and the samples were labeled with Cy3, and hybridized to human one array version 4.2 (HOA 4.2) DNA microarrays (Phalanx Biotech) containing 30,968 features probing for approximately 20,230 unique genes, according to standard procedures followed at the Functional Genomics of the Cancer Institute of New Jersey. Microarrays were scanned with the GenePix 4000B Scanner (Axon Instruments). The Gene Expression Omnibus (GEO) number for the microarray data is GSE49281.
Flow cytometry
Flourescence activated cell sorting (FACS) analysis was performed using a Beckman Coulter Epics XL. Cell sorting was performed using MoFlo XDP cells sorter (Beckman, Coulter). Cell lines stably transfected with expression vectors were sorted for cells with high expression of GFP or no GFP expression. Post sorting FACS analysis was used to confirm expression. For FACS analysis, the primary antibody mouse anti-human CD44 (BP Parmingen, San Diego, CA) was added to cells on ice for 60 minutes. Secondary antibodies were added for 60 minutes on ice. Control cells were treated with secondary antibody only.
Isolating cells from tumors
Tumor xenografts from mice were minced and treated with “digestion buffer” (10 ml HBSS, 50 mg collagenase powder, 200 μl 2.5% trypsin, 50 μl 1 M CaCl2, 50 μl DNAse). Fetal bovine serum (FBS) was added and samples were passed through a 100-micron filter. Cells were centrifuged and suspended in 3 ml of red blood cell lysis buffer (0.15 M ammonium chloride, 7 mM potassium bicarbonate, 0.09 mM tetrasodium EDTA) for 10 minutes. By FACS, the GFP (+) cells were then isolated.
Western blot analysis
Total cellular protein was prepared using RIPA buffer containing a protease inhibitor cocktail and protein concentration was measured using the BCA assay as described [
37]. In brief, protein was analyzed by SDS-PAGE, transferred to nitrocellulose (Schleicher and Schuell, Keene, NH). After blocking, the blots were incubated overnight at 4°C with the appropriate primary antibody in Tris-buffered saline with 1% Tween (TBST) and 5% non-fat milk. Secondary antibodies were applied for 1 hour at room temperature. Specific proteins were detected using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The primary antibodies that were used were rabbit monoclonal anti-pSmad 1/5/8 (Cell signaling Technology, Danvers MA) rabbit anti-actin, an affinity isolated antigen specific antibody (Sigma, Saint Louis, MO), rabbit monoclonal anti-Id1, rabbit monoclonal anti-Id3 (Calbioreagents, San Mateo, CA), rabbit anti-Oct 4 (Santa Cruz, Santa Cruz, CA), mouse anti-human nestin (Chemicon), and mouse anti-NeuN (Chemicon, Temecula, CA).
Differentiation of single cells
By FACS, the GFP (+) and GFP (−) cells were isolated from Oct4/GFP and Nestin/GFP cell lines and one-hundred cells placed into cloning chambers containing cell culture medium (Nunc Lab-Tek, Rochester, NY) [
33]. Cells were cultured in regular culture media for approximately 14 days until colonies formed. Immunofluorescent imaging was then performed as described above.
Statistical analysis
To compare two groups, a student t-test was used. Differences with P values ≤ .05 were considered statistically significant.
Discussion
CD133+ and CD44+ cells are reported to represent “cancer stem cells” in lung carcinomas, which have also been shown to express Oct4 and/or nestin [
11,
20,
21]. We provide evidence that lung cancer cells expressing Oct4 or nestin are different cell populations. The level of expression of nestin, BMP receptors, and other stem cell regulators are differentially expressed between the Oct4/GFP and Nestin/GFP cells. We also demonstrate biological differences between the Oct4/GFP and Nestin/GFP cells. The Nestin/GFP cells grew faster in nude mice than Oct4/GFP cells and form poorly differentiated tumors. The Oct4 cells formed more differentiated tumors and had a much large number of cells expressing smooth muscle actin. The response to BMP receptor antagonist also differed. DMH2 induced the expression of nestin in the Oct4/GFP + cells but not in the Nestin/GFP + cells. Inhibition of the BMP signaling cascade also caused more cell death in the Nestin/GFP cells compared to the Oct4/GFP cells.
We show that CD44 is expressed in nearly all cancer cells in our cell lines and CD133 is expressed in both Oct4 and nestin cell populations. Other reports have demonstrated that CD133 + cancer cells also express Oct4, nestin, nanog, and Sox2 [
1,
51]. The level of expression of Oct4 and/or nestin in cancer cells may induce specific survival mechanisms. Knockdown of nestin with siRNA decreases migration and invasiveness of pancreatic cancer cell lines [
52]. Nestin regulates survival and self-renewal of neural stem cells [
53]. Patients with NSCLC expressing nestin developed more metastasis and had a poorer survival [
41]. Knockdown of Oct4 with siRNA in CD133 + lung cancer cells induced apoptosis, decreased tumorigenicity, and increased sensitivity to chemotherapy and radiation [
20]. Our differentiation assays suggests that Oct4 cells give rise to cancer cells expressing nestin and NeuN. Further studies are needed to determine if a hierarchal organization occurs in “cancer stem cells” and examine the biology of other population of cells found within lung carcinomas.
BMP2 and BMP4 are highly conserved proteins required for development from insects to humans. BMP signaling is not active in adult lung tissue but is reactivated with inflammation and cancer [
54,
55]. BMP2 is highly overexpressed in 98% of NSCLC with little expression in paired normal lung tissue and benign lung tumors [
55]. BMP-2 signaling is associated with poor prognosis and tumor progression [
56,
57]. BMP signaling has been shown to stimulate cancer growth, survival, migration, invasion, metastasis, and tumor angiogenesis of several different tumors [
36,
37,
58‐
64]. We show that pharmacological blockade of BMP type I receptors causes significant growth inhibition of lung cancer cells expressing Oct4 or nestin. Inhibition of BMP signaling also caused significant growth inhibition and of non-selected cancer cells and GFP (−) cells, which were less tumorigenic. These data suggest that BMP antagonists affect the growth of more than just the Oct4 and nestin populations. Since cancer cells expressing stem cell makers represent only a small percentage of the cancer cells, therapeutically targeting the other cell populations is likely needed.
BMP receptor antagonists mediate growth inhibition of lung cancer cells by downregulating the expression of Id proteins [
32]. BMP2/4 stimulates self-renewal of embryonic stem cells by inducing the expression of Id1 [
29]. Studies have shown that Id1 mediates self-renewal of “cancer stem cells” and resistance to chemotherapy [
12,
13]. Within high grade gliomas, cancer cells with high Id expression (Id1-high) had a high self-renewal capacity [
12]. Cancer cells with low expression of Id1 (Id1 low) were highly proliferative with little ability to self-renewal [
12]. Inhibition of Id1 in Id1-high cells decreased self-renewal capacity and in Id1-low cells it decreased proliferation, suggesting that Id proteins have more than one biological function. Silencing of Id1 and Id3 together decreased self-renewal and increased sensitivity to chemotherapeutics of colon cancer-initiating cells [
14]. We show that DMH2, a small molecule antagonist of the BMP type I receptors, effectively decreases Id1 and Id3 expression in lung cells expressing stem cell markers. Future studies are needed to determine whether BMP antagonists enhance the effectiveness of chemotherapeutics and decreases self-renewal of cancer cells expressing stem cell markers.
Competing interests
A patent application was submitted for the use of BMP antagonists for the treatment of cancer. There have not been any royalties paid or anticipated in the near future regarding this work.
Authors’ contributions
EL carried out the molecular biology studies and assisted interpretation of the data. MD carried out and analyzed immunohistochemistry studies. EZ performed and interpreted microarray studies. JL planned experimental design, interpreted all data, and drafted manuscript. All authors read and approved the final manuscript.