THE ROLE OF MAP3K8 IN LUNG TUMORIGENESIS
A dissertation submitted to the
Division of Research and Advanced Studies Of the University of Cincinnati
In partial fulfillment of the requirements for the degree of
DOCTORATE OF PHILOSOPHY (Ph.D.)
in the Department of Environmental Health of the College of Medicine
December 3, 2003
by
Adam Michael Clark
B.S. University of Cincinnati, 1996
Dissertation Committee Marshall Anderson, Ph.D., Chair Jonathan S. Wiest, Ph.D. Alvaro Puga, Ph.D. Mario Medvedovik, Ph.D. Ranjan Deka, Ph.D. Linda Parysek, Ph.D. Abstract
The MAP3K8 (Cot/Tpl-2) protooncogene is a serine/threonine kinase that participates in the MAP kinase cascades (MEK-1, SEK-1, and MKK6), NFAT activation, the NF-kB signalsome, and Caspase-9 induced apoptosis. Activation of MAP3K8 is believed to contribute to cellular transformation and tumorigenesis. MAP3K8 was examined in lung cancer cells to determine if the gene played a role in lung tumorigenesis. Three areas related to tumorigenesis were examined: mutational analysis of the MAP3K8 gene in human lung cancer cells, expression analysis of MAP3K8 in lung cancer cell lines, and alterations of non-transformed lung cells upon transfection of
MAP3K8. PCR-based techniques (RT-PCR, SSCP, RACE, and Realtime PCR) analyzed mutational and expressional alterations of the gene. This was complemented with primer extension analysis to identify the promoter and Western blot analysis to confirm protein expression. Additional assays characterized alterations of lung cell activity including transfection, FACS analysis, Caspase activity analysis, and two different protein arrays.
In this thesis, the first mutation of MAP3K8 occurring in a primary human tumor was identified in a lung adenocarcinoma resulting from a rearrangement of the 3’end of the
RNA. Additional mutations were not found in either the 3’ end or open reading frame of the gene. Expression analysis demonstrated increased levels of MAP3K8 transcript in a large fraction of NSCLC cell lines, contrasting decreased levels of transcript in the majority of SCLC cell lines. These levels, however, did not correlate with protein expression in the cell lines examined. The transfection of an immortalized bronchial epithelial cell line (9HTE) with wildtype or mutant MAP3K8 plasmids slowed proliferation of the cells in a non-apoptotic manner compared to cells transfected with an empty vector. Examination of the activated pathways in the transfected cells identified altered activity of multiple transcription factors including NFAT, c-Myb, and Brn-3; and implicated a role for MAP3K8 in the mTOR and PKC signaling pathways. These data demonstrate that MAP3K8 is rarely mutated in lung tumors, but suggest that both altered transcriptional and translational regulation are associated with lung tumorigenesis. The transfection of MAP3K8 in non-transformed lung cells slowed proliferation, indicating that other molecular alterations are necessary to complement an oncogenic role for
MAP3K8 in lung cancer.
This dissertation is dedicated to my parents, Jim and Kathi, for their unconditional Love, Guidance, and Support, and to the kids; Allison, Cooper, and Rachael for keeping me young at heart 1
Table of Contents
1. Background………………………………………………………...………………4
I. Lung Cancer………………...……………………………………………...5
II. The MAP Kinase Signal Transduction Pathway……………..……….…..10
III. The MAP3K8 Protooncogene………………………………………….....15
2. Hypothesis and Specific Aims………………………………………………..…..31
3. Introduction……………………………………………..………………………...35
4. Manuscript 1: Mutational activation of the MAP3K8 protooncogene in lung
cancer…………………………………………………………..…………………………..38
5. Manuscript 2: Aberrant expression of the MAP3K8 protooncogene in lung cancer
cell lines and the effect of MAP3K8 activity in lung cells……………………..…62
6. MAP3K8 Expression in 9HTE Cells and the Effect on Downstream Pathways..107
7. Conclusions…………………….………………………………………………..137
8. Acknowledgements……………………………….……………………………..152 2
List of Table and Figures
Chapter 1 Figure 1. Cellular pathways in which MAP3K8 participates.
Chapter 2 Figure 1. The three areas of study.
Chapter 4 Figure 1. Southern blot analysis of the transforming gene. Figure 2. The MAP3K8 lung cancer mutant sequence. Figure 3. Scansite analysis of the wildtype and mutant MAP3K8 amino acid sequences. Figure 4. The MAP3K8 mutation exists in the primary lung adenocarcinoma L- 41. Figure 5. 3’RACE analysis of the MAP3K8 gene in lung cancer cell lines. Figure 6. SSCP analysis of the open reading frame of MAP3K8.
Table 1. MAP3K8 induced tumor formation in nude mice. Table 2. Primer pairs for MAP3K8 PCR-SSCP.
Chapter 5 Figure 1. 5’ RACE identifies the putative MAP3K8 promoter region and an alternatively spliced exon. Figure 2. Fluorescent labeled primer extension of the MAP3K8 RNA. Figure 3. Analysis of the MAP3K8 promoter region. Figure 4. PCR-SSCP of the MAP3K8 promoter. Figure 5. Realtime PCR of the MAP3K8 cDNA. Figure 6. MAP3K8 Western blot analysis of the lung cancer cell lines. Figure 7. Stable transfection of the MAP3K8 wildtype and mutant gene into 9HTE cells Figure 8. Expression of MAP3K8 wildtype and mutant genes slows 9HTE proliferation. Figure 9. MAP3K8 does not induce apoptosis in 9HTE cells Figure 10. 9HTE growth curve analysis 2
Table 1. Transcription initiation sites. Table 2. MAP3K8 promoter polymorphisms within lung cancer cell lines. Table 3. Comparison of MAP3K8 expression data Table 4. Cell cycle analysis of transfected 9HTE cells.
Chapter 6 Figure 1. Illustration of the TranSignal Protein/DNA procedure from Panomics Protocol. Figure 2. TranSignal Protein/DNA array. Figure 3. MAP3K8 TranSignal Array Analysis. 3
Figure 4. PCR amplification of the transcription factor regulated genes. Figure 5. Kinexus Phospho-Site Screen (KPSS-4.0) Analysis. Figure 6. Relative MAP3K8-induced Site-specific Phosphorylation. Figure 7. Illustration of the proposed MAP3K8-induced inhibition of the mTOR signaling pathway.
Table 1. Site-specific phosphorylation data.
Chapter 7 Figure 1. Expression level of MAP3K8 with the Affymetrix DNA microarray Figure 2. Mean value of MAP3K8 expression in the different tumor types
List of Abbreviations
ACS: American Cancer Society ATP: Adenosine Triphosphate BLAST: Basic Local Alignment Search Tool CMV: Cytomegalovirus COT: Cancer Osaka Thyroid EST: Ewing Sarcoma Transformant FACS: Fluorescence-Activated Cell Sorter GDP: Guanosine Diphosphate GTP: Guanosine Triphosphate LPS: Lipopolysaccharide MAPK: Mitogen Activated Protein Kinase MAP2K: Mitogen Activated Protein Kinase Kinase MAP3K: Mitogen Activated Protein Kinase Kinase Kinase MAP3K8: Mitogen Activated Protein Kinase Kinase Kinase 8 MTOR: Mammaliam Target of Rapamycin MUT: Mutant NFAT: Nuclear Factor of Activated T Cells NFKB: Nuclear Factor Kappa B NCI: National Cancer Institute NIH: National Institutes of Health NSCLC: Non-Small Cell Lung Cancer PCR: Polymerase Chain Reaction PTK: Protein Tyrosine Kinase RACE: Rapid Amplification of CDNA Ends RPTK: Receptor Protein Tyrosine Kinase SAPK: Stress Activated Protein Kinase SCLC: Small Cell Lung Cancer SSCP: Single Strand Conformation Polymorphism TPL-2: Tumor Progression Locus 2 TUNEL: Terminal UTP Nick End Labeling WT: Wildtype 4
Chapter 1
Background 5
I. Lung Cancer
Lung cancer is the leading cause of cancer related deaths in the United States with
171,900 new cases and 157,200 deaths estimated to occur during 2003 (American Cancer
Society, NCI SEER program). A relatively rare disease at the turn of the 20th century, lung cancer incidence increased in U.S. men ultimately peaking in 1984; after which lung cancer mortality rates began a slow decline. However, the incidence of lung cancer in
U.S. women has continued to increase, with more women dying now from lung cancer than breast cancer. Evidence is indicating that the incidence in U.S. women is beginning to plateau, but during 2003 lung cancer is still estimated to kill more people than the next four most common cancers (colo-rectal, breast, pancreas, and prostate) combined (ACS,
Cancer Facts and Figures, 2003).
Ironically, despite the high mortality rate, lung cancer is one of the most preventable diseases. Between 85- 90% of all new lung cancer cases are primarily caused by a single etiological source, tobacco smoke inhalation. Tobacco smoke is a complex carcinogenic agent composed of at least 69 animal carcinogens including: polycyclic aromatic hydrocarbons such as the human carcinogen benzo(a)pyrene; N-nitrosamines; aromatic amines; aldehydes; phenolytic compounds; volatile hydrocarbons, such as benzene; nitrohydrocarbons; and a variety of other miscellaneous organic and inorganic compounds, such as acrylamide and arsenic (Hoffmann et al., 2001). The various carcinogens include both tumor initiators and tumor promoters leading to tumorigenic development through a variety of mechanisms. For example, benzo(a)pyrene (BaP) is a known lung carcinogen that mutates DNA through the formation of DNA adducts, 6 primarily binding to a guanine base and resulting in a GC to TA mutation in the DNA sequence. Similarly, metabolites of the N-nitrosamine, NNK, mutates DNA by alkylating various positions of the DNA molecule resulting in a mixture of deoxyguanosine adducts (Pfeifer, et al., 2002). Metabolism of NNK also produces compounds that methylate DNA, a non-mutatgenic, epigenetic alteration that can result in changes in chromatin structure and protein-DNA interactions (Cloutier et al., 1999).
Compounds such as nicotine, which are themselves not considered to be carcinogenic, contribute to tumorigenesis through activation of the signal transduction pathways, such as MAP kinase and PKC growth pathways, and also through suppression of growth inhibitive pathways, such as modulation of the retinoic acid induced growth inhibitive pathway (Hecht, 1999; Chen et al., 2002; Schuller et al., 2003). Due to the abundance of carcinogens and co-carcinogens present in tobacco smoke, the combined mechanistic activity these compounds contribute to tumorigenic initiation and promotion is not completely understood, but the causal relationship between cigarette smoke inhalation and lung tumorigenesis is widely accepted.
There are many other risk factors in addition to cigarette smoke that contribute to progression of lung cancer, including: environmental (second-hand or passive) tobacco smoke, environmental arsenic, asbestos and other man-made vitreous fibers (MMVF’s), radon, and air pollution; all with different mechanisms of carcinogenic progression. For example, asbestos fibers are an independent lung carcinogen associated with an increase in lung tumor development. Asbestos exposure wounds lung tissue causing chronic inflammation in the lung that is believed to contribute to tumorigenic development.
Smokers have about a 10-fold increased risk of developing lung cancer compared to non- 7 smokers. This risk increases to over 50-fold in combination with asbestos exposure, an example of a multiplicative effect of the co-carcinogens. Radon exposure on the other hand, is more additive in its effect when associated with smoking-induced lung cancer.
Radon decay produces high linear energy transfer alpha particles depositing energy that produces reactive oxygen species (ROS) and oxidative stress within the cell (Alavanja,
2002; Alberg and Samet, 2003). ROS can alter and damage many molecules within the cell including lipids, nucleic acids, proteins and carbohydrates causing a synergistic effect in combination with other smoking-induced alterations.
The overwhelming majority of lung cancer tumorigenesis is associated with smoking, only about 20% of smokers develop lung cancer, suggesting that there is a genetic component associated with smoking-induced lung cancer. Across the worldwide population many genetic polymorphisms exist that alter the protein and affect the individual’s susceptibility to certain types of environmental insults and diseases. For example, polymorphisms exist within certain DNA repair genes such as XRCC1 (X-ray cross-complementing group 1) and ERCC2 (excision repair cross-complementing group
2). These polymorphisms influence the proteins’ activity in base-excision and nucleotide excision repair pathways that remove bulky DNA adducts. Carriers of these polymorphisms are high risk candidates for lung cancer development because they are more susceptible to DNA adduct-induced mutation from exposure to tobacco smoke
(Zhou et al., 2003; Kiyohara et al., 2002).
Polymorphisms in metabolic enzymes such as the phase I cytochrome P450s or the phase II glutathione S-transferase genes are associated with metabolism of xenobiotic compounds entering the cell. The phase I reactions are typically small molecule reactions 8 and include oxidation, reduction, and hydrolysis, while the phase II reactions are conjugation reactions such as glucuronide and sulfate conjugation of the xeniobiotic compound. Polymorphisms within these enzymes change the efficiency of conversion of a pro-carcinogenic compound to a carcinogenic compound. The roles of these metabolic enzymes and their effect on different compounds are still being investigated, but positive associations have been identified between the different polymorphic genotypes of the metabolic enzymes and lung cancer development (Kiyohara et al., 2002). While these polymorphisms are associated with susceptibility to smoking-induced lung tumorigenesis, other genes are activated or inactivated in the cell and affect growth, survival, and differentiation pathways and progressing the cell towards tumorigenesis.
The progression of a normal cell to a cancer cell is a multi-step process involving activation of oncogenes and inactivation of tumor suppressor genes. Lung cancer is no exception. Lung cancer is divided into two major histological types, small cell lung cancer (SCLC) and the non-small cell lung cancer (NSCLC). NSCLC is subdivided into squamous cell, large cell, and adenocarcinoma. These different histologies have different oncogenes and tumor suppressor genes that are targeted for alteration during tumorigenesis. One classic tumor suppressor pathway associated with lung tumorigenesis is the p16/Rb pathway regulating cell cycle progression. In SCLC cells, inactivation of
Rb through deletion, non-sense mutation, and splicing abnormalities occur in almost 90% of these tumors. In contrast, Rb inactivation is a rare occurrence in NSCLC. However, homozygous deletion, point mutations, and promoter hypermethylation of p16 occur in
30-70% of the NSCLC, but are relatively absent in SCLC (Sekido et al., 2003). The K-
Ras growth and proliferation signals are targeted in NSCLC, found in 20-30% of lung 9 adenocarcinomas and 15-20% of all NSCLCs, but are extremely rare in SCLCs. K-Ras point mutations constitutively activate the protein, stimulating the MAP kinase pathway; a pathway associated with cellular transformation and tumor progression (Sekido et al.,
2003). Within SCLCs, the autocrine signaling pathway activated by production of gastrin-releasing peptide (GRP) is expressed in 20-60% of the tumors, but are infrequently activated in NSCLCs even though these cells express the GRP receptor (Sekido et al.,
2003). Thus, the steps occurring during tumorigenic transformation of the different lung cancer types vary, but each has a positive influence on growth and proliferation and an inhibitive influence on growth suppression.
Interestingly, humanSCLCs have displayed opposite responses to alterations found in NSCLCs when transfected into the cells. As stated previously, K-Ras mutagenic activation stimulates the MAP kinase signal transduction pathway through activation of the map kinase kinase kinase, Raf-1. Similarly, activated Raf-1 is associated with cellular transformation like K-Ras. However transfection of activated K-Ras into small cell lung cancer cells resulted in decreased proliferation and a forty-fold decrease in cloning efficiency of the cells suggesting that activated K-Ras slows growth and decreases tumorigenic potential in SCLCs (Mabry et al., 1989). Transfection of an activated Raf-1 into SCLCs displayed almost identical results suppressing cell proliferation and cloning efficiency on soft agar. These cells also displayed a high level of phosphorylated MAPK accompanied by induction of the cyclin-dependent kinase inhibitor p27kip1, both of which were attenuated upon treatment of the MAP kinase kinase inhibitor PD098059 (Ravi et al., 1998, Ravi et al., 1999). These results suggest that activation of the MAP kinase pathway inhibits proliferation of SCLCs an activity that has been subsequently identified 10 as “dormant tumor suppressor pathways” (Weintraub, 1999). In Rb negative cell lines, activation of the pathways induces a growth arrest through increased synthesis and stability of the p27kip1 protein, an Rb independent cell cycle inhibitor. However, in Rb positive SCLC cells the p27kip1 protein level remained the same, but p16 protein levels increased activating Rb-induced growth arrest. The authors suggest that inappropriate growth signals are recognized by the cellular machinery and in response, activate the inactive, but intact tumor-suppressive pathways in the cell. The mechanisms for activation of these pathways is not well understood, but it is clear that different cellular contexts will induce different responses.
Lung cancer is a complex disease that has reached epidemic levels in the United
States. Progression of the disease stems from a variety of environmental health hazards, but primarily tobacco smoke inhalation. However, evidence indicates that certain genotypes may have a predisposition to developing lung tumors through the complex gene-environment interactions. Since multiple molecular alterations occur during lung tumorigenesis that influence the growth and metastatic potential of the cells, discovering the genes that are altered and understanding the influence they have of the cellular characteristics is essential for better diagnosis and treatment of lung cancer victims.
II. The MAP Kinase Signal Transduction Pathway
The MAP (Mitogen Activated Protein) Kinase signal transduction pathways are complex multiprotein pathways that are necessary to interpret signals received at the cell surface and initiate appropriate cellular response. MAP kinase pathways are three-tiered signaling cascades with protein activation events occurring through phosphorylation of 11 serines, threonines, and tyrosines. The basic structure of this pathway is conserved among eukaryotes from yeast to humans and involves the downstream activation of the MAP kinase kinase kinase (MAP3K) serine/ threonine kinases, to the downstream MAP kinase kinase (MAP2K) dual specificity tyrosine and threonine kinases, to a MAP Kinase
(MAPK). The MAPK translocates from the cytoplasm into the nucleus to elicit further molecular activities including phosphorylation of nuclear proteins and regulation of transcription factors (Garrington and Johnson, 1999; Pearson and Cobb, 2002; Widmann et al., 1999). In addition, other proteins such as GTP binding proteins and certain kinases that phosphorylate the MAP3Ks are considered to be MAP4Ks by certain groups. This linear downstream activation pathway is an oversimplification when considering there are multiple protein components at each tier and multiple phosphorylation sites on each protein. The pathways also have redundant activities causing an overlap among the different kinases that phosphorylate the same downstream protein. All these factors contribute to complex protein interactions within the cell and, in turn, elicit a wide variety of cellular activities.
Further complicating an understanding of the MAP kinase signal transduction pathways are the various activators of these pathways. Different transmembrane receptors, cellular stresses, and extracellular stimuli induce variability at the cell surface; in addition, there are multiple isoforms of the MAP kinase proteins with different activities and regulatory domains to increase the variation. These pathways have yet to be completely delineated from activation at the cell membrane to translocation into the nucleus. To date, at least fourteen MAP3Ks, seven MAP2Ks, and twelve MAPKs are known to exist in mammalian cells (Garrington and Johnson, 1999). The MAP3Ks tend 12 to be the most diverse and contain regulatory domains not found in MAP2Ks. The domains are necessary for cooperation with a variety of upstream inputs, scaffolding proteins, and anchoring proteins that determine the proper association and activation of the downstream MAP2Ks (Garrington and Johnson, 1999). On the other hand MAP2Ks tend to be highly specific in phosphorylation of MAPKs, but there is limited understanding of how these proteins integrate the activation signals for their downstream phosphorylation events (Pearson and Cobb, 2002). It is known that the different MAP2Ks recognize specific Thr-X-Tyr activation motifs on their downstream substrates based on the tertiary structure. Phosphorylation at these specific motifs is necessary to activate and regulate the downstream MAPK protein (Widmann et al.,1999). The MAPKs are the final kinase in the three-tiered pathway, but are not the final phosphorylation event to occur.
Activation of the MAPKs through phosphorylation of the Thr-X-Tyr motif induces nuclear translocation and phosphorylation of different MAPK substrates including transcription factors, protein kinases, phospholipases, and cytoskeletal associated proteins
(Widman et al., 1999)
Many studies have implicated activation of the MAP kinase pathways in cellular tumorigenesis. The receptor protein tyrosine kinases (RPTK) and cytoplasmic protein tyrosine kinases (PTK) utilize the MAP kinase transduction pathways to transmit signals from the plasma membrane into the nucleus. In these pathways, activation of the RPTKs or PTKs at the plasma membrane initiates recruitment of adaptor proteins, such as the
SH2 proteins, exchange factor proteins, such as Sos, and G-coupled proteins, such as Ras.
These multi-protein complexes activate the MAP kinase pathways (Hunter, 1997). The
RPTKs and PTKs have been associated with oncogenic transformation as transforming 13 retroviral oncogenes, genomic rearrangements resulting in oncogenic fusion proteins, gain of function mutants, and overexpressed and gene amplified proteins (Blume-Jensen and
Hunter, 2001). For example, the epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor protein tyrosine kinases. The EGFR is frequently overexpressed in the majority of NSCLCs and also produces truncated active forms, such as EGFRvIII, a variant form presumed to result from alternative splicing or rearrangement events (Tang et al., 2000; Bunn and Franklin, 2002). Activation of the EGFR affects multiple downstream pathways, including the MAP kinase pathway, through intricate multiprotein interactions. These multiprotein complexes lead to activation of Ras and the
“classic” MAP kinase pathway through Raf-1. This pathway induces expression of anti-
apoptotic proteins such as Bcl-XL and activates regulators of anti-apoptotic genes, such as the transcription factor CREB (Grant et al., 2002). Even though the RPTKs and PTKs are frequently targeted for oncogenic mutations, they are not considered part of the MAP kinase signal transduction cascades (Blume-Jensen and Hunter, 2001). However, these proteins play an integral role in activation of the cascades and utilize the MAP kinase pathways to convey signals regulating growth, proliferation, differentiation and apoptosis.
Other activators of the MAP kinase pathway have frequently occurring mutations in tumorigenesis. One well-characterized example is the oncogene K-Ras. The Ras proteins are active when bound to guanosine triphosphate (GTP) and are inactivated upon hydrolysis of the GTP to guanosine diphosphate (GDP). Activated Ras targets the MAP kinase pathway as one of its downstream factors. In NSCLCs mutations of the K-Ras gene occur frequently at codons 12, 13, and 61. These mutations prevent the hydrolysis of
GTP to GDP, resulting in a constitutively activated Ras protein, and, subsequently, an 14 activated MAP kinase pathway. In transformation assays, transfection of the wildtype c-
H-Ras under its own promoter was sufficient to transform the NIH3T3 cells. Upon co- transfection with a wildtype Raf-1, the transformation efficiency increased dramatically through increased activation of the MAP kinase pathway and the resultant c-jun driven transcription (Cuadrado et al., 1993). Thus, both mutated and over-expressed wildtype activators of the MAP kinase pathway are able to confer tumorigenic characteristics.
Conversely, frequent mutations of the MAP kinase signal transduction proteins are rare in human malignancies. This is in stark contrast to other proteins regulating growth, survival, and proliferation in other signaling pathways such as PI(3)K/Akt and mTOR/p70S6K , which display mutations in a variety of human malignancies (Blume-
Jensen and Hunter, 2001). It has been demonstrated that activating mutations for
MAP3K and MAP2K proteins can be designed in vitro to induce tumorigenic characteristics in cells, but mutational analysis of the MAP3K proteins, Raf-1 and TAK1, and the MAP2K proteins, MEK-1 and MEK-2, identified a very low occurrence of mutation or polymorphism of the genes in lung cancer cells (Welch et al., 2000; Bansal et al., 1997; Kondo, et al., 1998; Bosch, et al., 1997; Miwa et al., 1994). Even the B-Raf gene, a member of the Raf family that is frequently mutated in melanomas, colon cancer and ovarian cancer, displayed only a 3% occurrence in lung cancer cell lines and lung primary tumors (Davies et al., 2002; Brose et al., 2002).
Despite the lack of frequently occurring mutations of MAP kinase genes in lung cancer cells, other alterations of the genes occur influencing cellular growth characteristics. Both the MEK-1 and B-Raf genes were characterized as “candidate lung tumor progression genes” after it was discovered that these genes displayed an increased 15 transcriptional expression associated with lung tumor progression when compared with nearby normal lung tissue (Yao et al., 2002). Similarly, increased protein levels of Raf-1 and the phosphorylated downstream MAPK proteins, Erk-1 and Erk-2, are observed in initiated and promoted mouse lung tumors compared to the normal lung cells
(Ramakrishna et al., 2002). Lung cell targeted expression (under the lung cell specific
SP-C promoter) of the wildtype Raf-1, resulted in the formation of lung adenomas within
10 months of development in mice (Kerkoff et al., 2000). Taken together, these data continue to support transcriptional and translational alterations of signal transduction proteins as contributing factors in lung tumor progression.
The intricacies of the signal transduction pathways from the various activators at the cell surface to the differential gene regulation in the nucleus are currently being delineated. Understanding how a stimulus affects cellular activity through these pathways is essential in understanding their significance in cancer and in developing treatments for cells undergoing tumorigenic progression. Identifying effective inhibitors of these pathways may serve as therapeutic targets for cancer and other diseases.
III. The MAP3K8 Protooncogene
The MAP3K8 protooncogene (also known as Cot, Tpl2, and EST) is a MAP kinase kinase kinase serine/threonine kinase that functions on the same level as Raf-1.
The gene localizes to human chromosome 10p11.2 and has highly conserved homologues in the rat and mouse genome. Human MAP3K8 is composed of 9 exons, the second of which is alternatively spliced (Sanchez-Gongora et al., 2000). Exon 3 contains the two alternative translation initiation sites resulting in two protein isoforms of 467 and 438 16 amino acids in length, and referred to as MAP3K8-a and MAP3K8-b, respectively (Aoki et al., 1993, Widmann et al., 1999). In vitro translation of the MAP3K8 transcript demonstrated that both isoforms possess intrinsic autophosphorylation activity on their serine residues. Immunoprecipitation of cellular extracts followed by phosphoamino acid analysis demonstrated that the MAP3K8-a isoform was phosphorylated only at serines residues, but the MAP3K8-b isoform was phosphorylated at serine and threonine residues
(Aoki et al., 1993; Aoki et al., 1991). In addition, pulse-chase analysis of the stability of the proteins in cultured cells revealed that both isoforms had short half-lives with the half- life of the MAP3K8-a isoform at 10 minutes, compared to the MAP3K8-b isoform at 30 minutes (Aoki et al., 1993). The differences in the amino termini, the phosphorylated residues, and the half-lives of the two isoforms, suggest that differences also exist in the functional role of the isoforms within the cell.
MAP3K8 was originally cloned and identified from a cellular transforming assay for SHOK (Syrian Hamster Osaka-Kanazawa) cells, in an assay similar to the NIH3T3 transforming assay to detect and identify transforming oncogenes isolated from genomic
DNA (Miyoshi et al., 1991). The SHOK cells were transfected with DNA from a human thyroid carcinoma. Upon transformation, the DNA was isolated and the transforming gene was originally named Cot (Cancer Osaka Thyroid), and later given the name
MAP3K8 through the accepted nomeclature. Sequencing analysis demonstrated that the gene was similar to other known serine protein kinases. Retransfection of the MAP3K8 cDNA into SHOK and NIH3T3 cells transformed both the SHOK and NIH3T3 cells at equal efficiency and subsequent kinase assays confirmed that the gene was a protein kinase (Miyoshi et al., 1991; Aoki et al., 1991). However, the authors speculated that the 17 isolated cDNA sequence had an altered 3’ end that resulted in an altered carboxy terminus of the transforming protein. Upon further inspection, the altered cDNA sequence could not be found in the original thyroid tumor DNA, suggesting that the alteration affecting the 3’end occurred during the transfection procedure as an experimental artifact (Miyoshi et al., 1991).
Shortly thereafter, a similar procedure isolated and identified cellular transforming genes through expression cDNA cloning of the genes into NIH3T3 cells. These cells were transfected with clones from a cDNA library constructed from poly(A) RNAs from a human Ewing sarcoma cell line. One of the transforming genes was named Est (Ewing
Sarcoma Transformant) and was identified by a literature search as an almost identical gene to Cot/MAP3K8 (Chan, et al., 1993). The Est and Cot genes differed in the 3’ end of the RNA and carboxy terminal amino acid sequence. It was discovered that the Est gene was the wildtype MAP3K8 gene encoding a protein of 467 amino acids, compared to the mutated MAP3K8 gene that coded a protein of 415 amino acids (Aoki, et al., 1993).
The mutated gene displayed higher transforming efficiency than the wildtype gene, suggesting that the carboxy terminal domain may negatively regulate the transforming activity of the protein (Aoki et al., 1993).
Identification of the rat homologue of MAP3K8, Tpl2 (Tumor Progression Locus
2), confirmed that loss of the carboxy terminus does activate the protein and increases the tumorigenic potential. The rat MAP3K8 gene is activated by proviral insertion of MMLV
(Moloney Murine Leukemia Virus) into the last intron of the gene leading to an altered mRNA transcript (Makris et al., 1993). This transcript produces a protein with an altered and shortened carboxy terminus that constitutively activates the kinase activity of the 18 protein leading to increased phosphorylation downstream of Erk1 and Jnk1 proteins (Ceci et al., 1997). Co-transfection of the mutated rat MAP3K8 with a fusion protein containing the wildtype carboxy terminal amino acids decreased this kinase activity suggesting that the carboxy terminus physically inhibits the protein activity (Ceci et al.,
1997). Mice expressing the mutated rat MAP3K8 gene developed large-cell lymphoblastic lymphomas, an occurrence not observed in mice expressing the wildtype gene (Ceci et al., 1997). These data, along with the data from the human MAP3K8 gene, suggested that though the wildtype MAP3K8 possesses a low level of tumorigenic potential through increased activation of MAP kinase pathways, but oncogenic activation of the gene occurs through loss of the carboxy terminus, releasing inhibition of the intrinsic kinase activity of the protein.
Subsequent studies of the gene revealed that it functioned on the level of the MAP kinase kinase kinases. The assorted names were thus afforded the nomenclature of
MAP3K8 in reference to the human, rat, and mouse homologues. The kinase domain of
MAP3K8 was homologous to the S. cerevisae MAP kinase kinase kinase, STE11, suggesting the gene may have a similar function (Salmeron et al., 1996). In vitro studies demonstrated that the MAP3K8 protein did indeed phosphorylate and activate the
MAP2Ks, MEK-1 and SEK-1, and transfection of the gene into cellular systems induced phosphorylation and activation of the MAP kinases Erk1 and Jnk1 in a variety of cell types including COS-1, Jurkat T, HEK-293, NIH3T3, and PC12 cells without the need for an activating extracellular stimulus (Patriotis et al., 1994; Hagemann et al., 1999;
Salmeron et al., 1996). Transfection of MAP3K8 into NIH3T3 cells activated additional
MAP kinase pathways through phosphorylation and activation of the MAP2Ks, MEK-5 19 and MKK6, along with the MEK-1 and SEK-1 activation. These converged through the
MAPKs to stimulate the c-jun promoter and transform the cells (Chariello et al., 2000).
Other results demonstrated that under certain conditions MAP3K8 may participate in multiprotein complexes with Ras and Raf to activate the downstream pathways. However, conflicting reports suggested that the proteins instead competed for the same downstream targets instead of actually interacting with each other (Patriotis et al., 1994; Salmeron et al., 1996). All of this indicates that MAP3K8 has a wide variety of downstream targets overlapping with other known MAP3Ks through complex, redundant signaling cascades.
Overexpression of the gene was sufficient to induce activation of the pathways in these different cell types. It remains unclear what activates and regulates MAP3K8 and induces appropriate cellular responses. In vitro translation studies indicate that intrinsic
MAP3K8 kinase activity is active upon translation of the protein without the necessity for an upstream stimulus. This intrinsic activity was confirmed to be inhibited by antibodies against the MAP3K8 ATP binding region, but not by antibodies against other regions of the protein (Aoki et al., 1993). However, signals transduced by members of the TNF receptor superfamily utilize MAP3K8 for activation of MEK-1/Erk1 pathway without having any effect on the SEK-1/Jnk1 or MKK6/p38 pathways (Eliopoulus et al., 2003).
Additionally, hKSR-2, a protein proposed to function as a scaffolding protein, interacts with and selectively inhibits MAP3K8-mediated MAP kinase activity when co-expressed with MAP3K8, but has no effect on other MAP3Ks and their downstream pathways
(Channavajhala et al., 2003). These data indicate that multiple factors including transcription and translation regulation, activation of upstream receptors, and binding to other proteins influence not only the intrinsic MAP3K8 kinase activity but also the 20 specificity of activation of the downstream pathways. Despite the vast amount of information accumulating on the MAP kinase pathways, the multiprotein complexes formed and the mechanisms inducing activation of their pathways are still not well understood.
Figure 1. Cellular pathways in which MAP3K8 participates. Four pathways are known to be involved with MAP3K8 activity. Clockwise starting at the top, these pathways are the MAP Kinase Signal Transduction Pathways, Cell Cycle regulation, Caspase-9 induced apoptosis, and NF-kB activation and the NF-kB Signalsome.
Further complicating the role the signaling proteins play in the cell is the dilemma that many of these kinases participate in other intracellular activities outside of the MAP kinase pathways, either directly or indirectly. MAP3K8 is no exception. In addition to 21 the MAP kinase pathways, MAP3K8 participates in cell cycle regulation, Caspase-9 induced apoptosis, induction of Nuclear Factor of Activated T cells (NFAT), and NF-kB signalsome activity. Similar to the identification of the downstream MAP kinase pathways, these cellular pathways were identified through transfection of the MAP3K8 gene into various cellular systems.
The alteration of cell cycle progression was originally identified upon transfection of the MAP3K8 gene into a T cell lymphoblast cell line. Expression of the gene correlated with reduced levels of the cycling kinase inhibitor p27kip and increased levels of
E2F transcription activity. Increased DNA synthesis and a higher percentage of cells reflected this in the G2/M phases of the cell cycle, although no direct relationship was identified linking MAP3K8 with regulators of the cell cycle (Valesco-Sampayo et al.,
2001). In contrast to this, transfection of both the wildtype and oncogenic MAP3K8 gene into mouse embryonic fibroblast (MEF) cells decreased proliferation to similar degrees.
However, knocking-out of the cdkn2a locus, the locus that transcribes the tumor suppressor p16, resulted in increase proliferation of the cells upon transfection of the
MAP3K8 gene (Lund et al., 2002). This suggests that different cellular contexts initiate different responses to MAP3K8 and that inactivation of tumor suppressors such as p16 are necessary for MAP3K8 to exert its proliferative effects.
In HEK293 and REF52 non-transformed fibroblast cells, transcription of MAP3K8 under an inducible promoter resulted in increased apoptosis of the cells (Patriotis et al.,
2001). MAP3K8 protein formed a multiprotein complex with the pro-apoptotic proteins
Pro-Caspase-9, Tvl-1, and Apaf. MAP3K8 phosphorylates Pro-Caspase-9, resulting in cleavage to Caspase-9. Caspase-9 recruits Pro-Caspase-3, processes it into Caspase-3, 22 initiating cellular apoptosis. This activity appears contradictive to a role in cellular oncogenesis, and, like the inhibition of cell cycle progression in MEF cells, may require certain cellular contexts and molecular participants to induce this activity.
NFAT is a family of four different transcription factors (NFATp, NFATc, NFAT3, and NFATx) encoded by different genes that are expressed in a variety of cell types, but integral in T-cell activation, regulation of the immune response, and expression of various cytokines, interleukins, and other early response genes. NFAT activity is linked with various cellular activities related to proliferation, survival, and apoptosis and is a significant protein in tumorigenesis. The NFAT proteins are activated by phosphorylation, and, in turn, translocate to the nucleus, binding to their DNA response elements to induce transcription of a battery of genes. Expression of MAP3K8 results in increased activation of NFAT and transcription of NFAT-responsive genes, such as IL-2 and COX-2 (Tsatsanis et al., 1998; Tsatsanis et al., 1998; de Gregorio et al., 2001).
Activation of NFAT reporter constructs was higher when transfected with the oncogenic activated MAP3K8 than wildtype MAP3K8. Both the wildtype and mutant MAP3K8- induced activation of NFAT were inhibited when the cells were treated with a
MEK1/MEK2 inhibitor, PD098059, indicating that the NFAT activation is instituted through the MAP kinase pathway (Tsatsanis et al., 1998)
Activation of the transcription factor NF-kB is an important activity regulating cytokine expression, cell adhesion, cellular survival and resistance to apoptosis. There are five members of the NF-kB family, all of which exist in most cell types, but require induction from upstream signals for activation. Two members, p105 and p100, are proteolytically processed into p50 and p52, respectively. This processing is required for 23 nuclear translocation and transcriptional activation. In addition the other members, p65
(RelA), RelB, and c-Rel are participants in the NF-kB signalsome where they are bound and inhibited by the IkB protein. The p105 and p100 precursor proteins contain ankyrin repeats near their carboxy terminus where other NF-kB proteins are bound. Proteolytic cleavage of p105 and p100 releases these bound proteins in addition to processing the p105 and p100 proteins into p50 and p52 allowing the processed proteins to translocate into the nucleus. Activation of the NF-kB proteins bound in the NF-kB signalsome occurs by a different mechanism. The nuclear translocation signal of the NF-kB proteins are masked by the IkB family of proteins. These proteins disassociate upon phosphorylation by the IkB Kinase (IKK) or other upstream kinases such as p90RSK. Additionally, proteins such as NF-kB Inducing Kinase (NIK) and MEKK-1 are believed to function upstream or in a multi-protein complex with the NF-kB signalsome to induce activation of this pathway through a variety of extracellular signals (Review by Ghosh et al., 1998).
MAP3K8 is able to induce activation of the NF-kB proteins through both the p105 and NF-kB signalsome pathways. To date no relationship has been found to exist between
MAP3K8 and p100/p52. MAP3K8 binds to p105 at the ankyrin repeats along with the p50 and p65 proteins. This binding stabilizes the MAP3K8 protein, but also inactivates it so that it is not able to activate the downstream signal cascades (Waterfield et al., 2003).
Release of MAP3K8 from p105 occurs by a mechanism that is not yet known. This release activates the MAP3K8 protein, which in turn, phosphorylates p105, causing release and nuclear translocation of p50 and p65 proteins and proteolytic degradation of p105. However, the signal transmitted from MAP3K8 does not induce proteolytic 24 processing of p105 to p50. Unbound MAP3K8 protein activates the MAP kinase pathway, suggesting that p105 functions as a MAP3K8 inhibitor.
Within the NF-kB signalsome MAP3K8 is believed to function in a multiprotein complex to induce activation of this pathway. Phosphorylation of MAP3K8 at serines
400 and 413 by Akt activates the protein, which, in turn, signals through NIK and causing the release and translocation of the p65 and p50 NF-kB proteins to the nucleus (Kane et al., 2002). The tumor suppressor PTEN, a phosphotase that regulates Akt activation, inhibits this pathway. It is not yet known what different factors contribute to MAP3K8 participation in these different pathways, but due to the variety of participants it is possible that MAP3K8s role changes in different cell types as well as by activation by different signals.
In summary, the MAP3K8 protein has many activities that can contribute to tumorigenesis outside of its role as a traditional MAP3K. It is not understood what factors regulate these activities, but loss of different tumor suppressors appears to be significant in MAP3K8s regulation of cell proliferation and activation of the NF-kB signalsome.
Similarly, the activation of the Caspase-9 induced apoptosis was not observed in tumor cells but in non-transformed cells, a cell system in which the cells are immortalized and do not have a finite number of cell divisions, but act more like normal cells than tumor cells. The NIH3T3 and SHOK cells, on the other hand, are cell systems that have almost progressed to a transformed cell and require an additional stimulus to fully transform. This progression allows genes like MAP3K8 to exert oncogenic properties and activate transformation pathways upon loss of the negative regulation of tumor suppressor genes. Also, data shows that increased MAP3K8 mRNA levels have been associated with 25 breast cancer, and induced expression of both wildtype and mutant MAP3K8 can transform different cell types (Sourvinos et al., 1999). All this taken together suggests that MAP3K8 is an excellent candidate oncogene for research in different tumors.
The recommendations of the Lung Cancer Progress Review Group at the National
Cancer Institute comprised three aims for the ongoing research of lung cancer in an effort to stop this epidemic. First, to perform basic biological research aimed at understanding the cellular and molecular biology attendant to each of the major forms of lung cancer.
Second, to explore signal transduction events that program the cells, including signaling to and from non-epithelial components of normal and adult lung, and relate these discoveries to steps in lung carcinogenesis. And third, to define the molecular switches of human lung cancer, clarify existing knowledge of lung cancer, and develop new knowledge of pathways underlying the development of normal and neoplastic bronchial epithelium
(Progress Review Groups, NCI, 2001). Understanding the role of MAP3K8 in lung cell proliferation, growth, development, and survival contributes to each of these three aims as well as contributing to our expanding knowledge of the functions of signal transduction proteins in tumorigenesis and environmental health. 26
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Chapter 2
Hypothesis and Specific Aims 32
The current hypothesis driving this research is:
Oncogenic alterations of the MAP3K8 protooncogene occur during lung tumorigenesis to influence cellular growth characteristics.
The initial research surrounding this hypothesis examined mutational activation of the gene. Since altered expression was discovered to be directly associated with breast tumors and increased expression of the wildtype gene conferred a transformed phenotype in a variety of cell lines, oncogenic alterations that influence MAP3K8 expression were also examined.
Figure 1. The three areas of study. Three different areas of MAP3K8 biology will be examined in this dissertation to test the hypothesis and demonstrate a role for MAP3K8 in lung tumorigenesis. 33
In order to test this hypothesis, three specific Aims were developed along with the methods to perform the tests:
1. Determine if MAP3K8 mutation is a frequent occurrence in lung cancer.
I. PCR amplification to confirm the existence of a MAP3K8 mutation in
a primary lung tumor
II. 3’RACE analysis to identify 3’end mutations in the mRNA of various
lung cancer cell lines
III. PCR-SSCP analysis to search for point mutations within the open
reading frame of MAP3K8 in various lung cancer cell lines
2. Examine alterations of transcription and translation of MAP3K8 in lung cancer
cell lines.
I. 5’ RACE, Primer Extension, and PCR-SSCP to identify the MAP3K8
promoter and any polymorphisms within the region that may be related
to expression
II. Realtime PCR to quantify MAP3K8 mRNA expression in various cell
lines and determine if a difference exists among the different types of
lung cancer
III. Western blot analysis to examine MAP3K8 protein expression levels
3. Examine whether transfected MAP3K8 affects lung cell growth characteristics.
I. Stably transfect wildtype and mutant MAP3K8 cDNA under the CMV
promoter into the immortalized bronchial epithelial cell line, 9HTE 34
II. Determine proliferation rates of the transfected 9HTE cells through a
growth curve analysis
III. FACS analysis of the transfected cells to analyze alterations within the
cell cycle
IV. TUNEL assay and a Homogenous Caspase Assay of the transfected cell
lines to determine if the changes in apoptosis or Caspase activity occur
V. Transcription factor array to determine the effect MAP3K8 has on the
activity of downstream transcription factors
VI. Phospho-protein array analysis to determine the phospho-status of
proteins downstream of the MAP3K8 protein 35
Chapter 3
Introduction 36
The project for this dissertation was developed from existing data published in a poster entitled “ Characterization of a Novel Proto-Oncogene in Human Lung Tumors from Cigarette Smokers” (Wiest J., Solomon G., Haugen-Strauno A., and Anderson M.
(1993). National Institutes of Environmental Health Sciences). The data presented in the poster identified a mutated MAP3K8 protooncogene that was isolated as a transforming gene from a human lung adenocarcinoma through the nude mouse tumorigenicity assay.
The discovery of a mutated MAP3K8 drove the original concept that MAP3K8 mutation was a targeted alteration in lung tumorigenesis, but also supported a broader hypothesis that MAP3K8 activity was important in lung tumorigenesis. It was at this point that I began research for my dissertation using the data presented in the poster as the foundation for my doctoral thesis. Since no mutation of MAP3K8 had been found to occur in a primary human tumor, but had been identified in rat tumors, I began trying to determine if the mutation identified from the lung adenocarcinoma occurred in the primary tumor
DNA.
The thesis will be divided between the two papers written during the course of this dissertation and include additional unpublished data that has been gathered in support of the hypothesis. The first paper will include data that I did not acquire but was provided in the aforementioned poster by Wiest et al., but is included in the dissertation because it is essential to the project, hypothesis, experimental reasoning, and conclusions generated.
The submitted papers will provide the data and results for the identification of the
MAP3K8 mutation, search for additional mutations, identification and characterization of the MAP3K8 promoter, expression characteristics of MAP3K8 mRNA and protein, and 37 alterations of the MAP3K8 proliferation characteristics. Additional data will demonstrate alterations among the molecular biology of lung cells due to MAP3K8 expression at both the level of the activated transcription factors and phosphorylated proteins. 38
Chapter 4
Manuscript 1
Mutational activation of the MAP3K8 protooncogene in lung
cancer 39
Title:
Mutational activation of the MAP3K8 protooncogene in lung cancer.
Authors:
1. Clark AM. Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for
Cancer Research, National Cancer Institute, Bethesda, MD. 20892,USA.*
2. Reynolds S. National Institute for Occupational Safety and Health, Morgantown, WV.
26505, USA
3. Anderson M. Department of Environmental Health, College of Medicine, University of
Cincinnati, Cincinnati, OH. 45267, USA
4. Wiest JS. Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for
Cancer Research, National Cancer Institute, Bethesda, MD. 20892,USA.*
Abstract
The MAP3K8 protoonocogene (Cot/Tpl-2) activates MAP kinase, SAP kinase, and the
NF-kB signaling pathways. MAP3K8 mutations occur in the rat homologue, but activating mutations have yet to be identified in primary human tumors. Previously, we reported a transforming gene in a human lung adenocarcinoma and showed it was
MAP3K8. In this report, we examined if the mutation occurred in the original lung tumor, and we screened a series of lung cancer cell lines to determine if MAP3K8 mutation is a 40 common occurrence in lung tumorigenesis. The NIH3T3 nude mouse tumorigenicity assay and subsequent cDNA library screening isolated and identified the oncogene. The gene was analyzed by PCR, SSCP, and 3’RACE for mutations. The mutation was identified in the 3’end of the MAP3K8 transcript and confirmed in the primary human tumor DNA. Both wildtype and mutant MAP3K8 genes transformed NIH3T3 cells, but the transforming activity of the mutant was much greater than that of the wildtype.
Screening of the cell lines identified one silent polymorphism in SK-LU-1. While we did not find additional 3’end alterations, these data support a role for MAP3K8 activity in cellular transformation but suggest that mutational activation of the gene is a rare event in lung cancer. 41
Introduction
Lung cancer is the leading cause of cancer related deaths in the US for both men and women. The initiation and promotion of a normal lung cell to a cancer cell is a multistep process involving alterations of genes regulating cellular growth, proliferation, and survival (Sekido et al., 1998). The MAP kinase signaling cascades and the NF-kB signalsome are key participants in these processes. MAP kinases transfer signals received at the cell surface from assorted receptors and G proteins through an intricate network leading into the nucleus to initiate a variety of cellular responses. Likewise, the NF-kB signalsome is composed of a multiprotein network that induces translocation of the NF-kB transcription factors into the nucleus influencing cytokine production, anti-apoptotic pathways, cellular differentiation, and production of cellular adhesion molecules (Ghosh et al., 1998). In lung cells, activation of these pathways plays a role in tumorigenesis, metastasis, and resistance to chemotherapeutic treatment (Brognard and Dennis, 2002;
Jones et al., 2000).
The MAP3K8 proto-oncogene (also known as Cot and Tpl-2) is a serine/threonine kinase that participates in MEK-1, MKK6, SAPK, NFAT and NF-kB signaling pathways
(Aoki et al., 1991; Belich et al., 1999; Chariello et al., 2000; Hagemann et al., 1999;
Patriotis et al., 1994; Salmeron et al., 19966; Tsatsanis et al., 1998(1); Tsatsanis et al.,
1998(2)). Transcriptional overexpression, mutational activation, and extracellular stimulation through LPS (Lipopolysaccharide), LMP1 (Latent Membrane Protein 1), and
CD3/CD28 influence MAP3K8-specific regulation of these downstream cascades 42
(Dumitru et al., 2000; Eliopoulis et al., 2002; Lin et al., 1999). Overexpression and mutation of MAP3K8 are implicated in a variety of tumors including thymomas, lymphomas, breast cancer, Hodgkins disease, and nasopharyngeal carcinoma, suggesting that altered activation of the gene is significant in tumorigenesis (Ceci et al., 1997;
Eliopoulos et al., 2002; Patriotis et al., 1993; Sourvinos et al., 1999).
The MAP3K8 gene was originally identified in human thyroid tumor DNA as a gene capable of transforming SHOK (Syrian Hamster Osaka Kanazawa) cells (Miyoshi et al., 1991). It was later discovered that the transforming gene contained a carboxy- terminal mutation of the MAP3K8 gene (Chan et al., 1993). However, this mutation was not found in the original thyroid tumor DNA, suggesting that it occurred as an artifact during the transfection procedure (Miyoshi et al., 1991). In contrast, the rat homologue,
Tpl-2, is frequently mutated in tumors by integration of Moloney Murine Leukemia Virus into the last intron of the gene, thus altering the 3’end of the transcript and the subsequent carboxy-terminus (Patriotis et al., 1993). The alterations found from both the human thyroid tumor DNA and the rat MAP3K8 oncogenes result in increased kinase activity and a higher transforming ability (Ceci et al., 1997; Hagemann et al., 1999).
Here we report the first known mutation of the human MAP3K8 gene occurring in a human primary tumor. We originally identified the MAP3K8 oncogene as an unknown transforming gene isolated from a lung adenocarcinoma (Reynolds et al., 1991). DNA sequencing revealed an altered 3’ end of the MAP3K8 transcript and PCR amplification of the original tumor DNA and subsequent sequencing of the amplicon confirmed that the mutation resided in the primary tumor. Transfection of the MAP3K8 wildtype and mutant cDNAs into NIH3T3 cells induced tumorigenic growth in nude mice and demonstrated 43 that MAP3K8 was the transforming gene isolated from the original transfection, paralleling the research by other groups that identified MAP3K8 as an oncogene (Ceci et al., 1997; Chan et al., 1993; Miyoshi et al., 1991). In order to search for additional mutations, we screened a variety of lung cancer cell lines by PCR-SSCP (Single Strand
Conformational Polymorphism) analysis for point mutations and examined alterations occurring in the 3’ end of the transcript by 3’RACE. Of the cell lines examined, no additional 3’ alterations were identified, but the SK-LU-1 cell line harbored a mutation/polymorphism in exon 7 of the coding region. While activation of MAP3K8 plays a role in NIH3T3 transformation, mutational activation of the gene appears to be rare event in lung cancer.
Materials and Methods
Identification of the Transforming Gene: The nude mouse tumorigenicity assay was performed as described (Fasano, 1984). In brief, high molecular weight DNA was isolated from a human lung adenocarcinoma, transfected into NIH3T3 cells, selected for positive transfection and colony formation, and subcutaneously injected into nude mice.
The resultant tumors were probed for human Alu positive sequences by Southern Blot, cloned into the lgt10 vector, and hybridized to a zoo blot. Of the Alu positive tumors, lgt11 cDNA libraries were made, screened for the human sequence, sequenced, and
BLAST searched identifying MAP3K8 transcript. To ensure that MAP3K8 was the gene responsible for inducing cellular transformation, NIH3T3 cells were re-transfected with the cDNA transcripts of the MAP3K8 gene and assayed for tumor formation in nude mice. 44
Cell Lines: The following lung cancer cell lines were grown in RPMI 1640 with 10%
FBS: H28, H69, H125, H157, H187, H324, H345, H372, H378, H446, H460, H526,
H661, H719, H720, H726, H727, H740, H865, H1048, H1062, H1334, H1581, H1607,
H2122, N417, A549, SHP77, NE-18, CAlu1, CAlu3, H322, CHAGO-K-1, CAlu6, SK-
LU-1, H441, NU-6-1, SW900, SK-MES-1, H290. The 9HTE cell line was grown in
DMEM with 10% FBS.
PCR Amplification: Products were amplified from lung cancer cell line cDNAs according to standard procedures. The mutation from lung tumor #41 was amplified from genomic tumor DNA with one MAP3K8 gene specific primer (3PF: 5’-
CCCTGGAGAGAAACCCCAATCACC-3’) and one mutation specific primer (MUT-
UX5: 5’-TGACAGGAGTAGACCAAATTGAA-3’).
Single Strand Conformational Polymorphism (SSCP) analysis: The primers for the PCR-
SSCP produced overlapping fragments spanning the open reading frame of the MAP3K8
(Table 2). cDNA was amplified by PCR, run in a 0.5X MDE polyacrylamide sequencing gels with and without 5% glycerol (Cambrex Biosciences, Rockland, Maine) and silver stained to view the bands (Promega, Madison, WI). Bands with altered migrations were sequenced to confirm polymorphic DNA sequences.
3’RACE: SMART RACE cDNAs (BD Biosciences Clontech, Palo Alto, CA) were made according to the manufacturers instructions. Nested primers were used to increase 45 sensitivity and specificity. The first round of amplification was performed with the
MAP3K8-2ACF-RACE (5’-CGATGAGCGTTCTAAGTCTCTGCTGC-3’) primer, followed by a second round of amplifications with the MAP3K8-5ACF (5’-
CAAAGCAGACATCTACAGCC-3’) primer and the RACE primers.
Computer Analysis: BLAST analysis was performed through the National Institute of
Health program at http://www.ncbi.nlm.nih.gov/BLAST/ and the Ensembl program http://www.ensembl.org. Amino acid analysis was performed with the Scansite program http://scansite.mit.edu.
Results
Identification of a novel MAP3K8 transforming mutation from a lung adenocarcinoma
The NIH3T3 nude mouse tumorigenicity assay was designed to identify transforming genes in genomic DNA from tumors (Fasano et al., 1984). NIH3T3 cells were transfected with genomic DNA isolated from the lung adenocarcinoma, L-41, subcutaneously injected into nude mice, and assayed for tumor formation. Southern blots probed with human Alu repeats confirmed the presence of human DNA in the mouse tumors. DNA from the nude mouse tumors was re-assayed to enhance selection of the transforming gene (Figure 1a). 46
a. b. Figure 1. Southern blot analysis of the transforming gene. a) Nude mouse tumor DNA was digested with EcoR I (left lane) or EcoR I and BamH I (right lane) and probed at high stringency with the human Alu sequence confirming the presence of human tumor DNA in the mouse tumor. b) Zoo blot probed with the human transforming sequence cloned into the pBluescript vector. Nude mouse tumor DNA (NMT #41) was used as a positive control. The probe hybridized at a molecular weight identical to the human DNA in lane 2 (Human T24) and to primate DNA in the subsequent lanes demonstrating the sequence is conserved, suggesting the probe contains an exon.
Nude mouse tumor genomic DNA was digested with EcoR I and cloned into lgt10. The human Alu element was used to screen plaques and three positive lgt10 clones were identified. These were subcloned into the plasmid vector pBluescript (data not shown). Alu positive plasmid clones were digested with BamH I and Alu negative fragments were identified by Southern blot and subcloned. The Alu negative clones were hybridized to a zoo blot to identify cloned sequences conserved across species that 47 potentially contained exons (Figure 1b). A zoo blot positive clone was used to screen lgt11 cDNA libraries prepared from the nude mouse tumors and a normal human cDNA library to isolate the transforming and wildtype genes. Sequence analysis and BLAST searches confirmed the transforming gene was the human MAP3K8. However, the cDNA isolated from the tumor library displayed a novel mutation in the transcript near the 3’ end of the open reading frame.
a. b.
Figure 2. The MAP3K8 lung cancer mutant sequence. a) Partial cDNA sequence of the MAP3K8 lung cancer mutation with the deduced amino acid sequence below. The bold capital A denotes the start of the penultimate exon of MAP3K8. The arrow indicates the nucleotide where the thyroid tumor mutation starts. The underlined sequence corresponds to the mutation sequence from the lung tumor. b) Ensembl BLAST analysis demonstrates that the wildtype MAP3K8 sequence (top) localizes only to chromosome 10, while the mutant sequence (bottom) localizes to chromosome 9 and chromosome 10. Red boxes indicate the region of highest homology to the genomic DNA. The red arrowhead indicates a high scoring region and the blue arrowhead indicates a low scoring region. The MAP3K8 mutant sequence has two red arrows due to the MAP3K8 wildtype sequence homology and the chromosome 9 homology contained in the mutation site. 48
Human MAP3K8 is located at chromosome 10p11.2. Figure 2a illustrates the nucleotide and amino acid sequence of the mutant MAP3K8. A BLAST homology search with the mutant sequence against the Ensembl database confirmed the mutation occurred as a genetic rearrangement/recombination of the penultimate exon of MAP3K8 with a sequence from chromosome 9. The wildtype sequence localized with high homology only to chromosome 10, while the mutant mapped two regions of high homology, the chromosome 10/MAP3K8 region and a chromosome 9 specific region from the 3’end of the sequence (Figure 2b).
Wildtype MAP3K8 produced a protein of 467 amino acids and the human thyroid tumor mutation resulted in a protein of 415 amino acids and an altered carboxy terminus
(Miyoshi et al., 1991). The lung tumor mutation we identified resulted in a putative protein of 429 amino acids with the first 421 identical to the wildtype and the last 8 novel to the sequence. Wildtype MAP3K8, lung mutation, and thyroid mutation amino acid sequences were analyzed with the Scansite program under medium stringency conditions to characterize differences in the sequences (Figure 3). The program identified three putative serine/threonine phosphorylation sites within the carboxy terminus of the wildtype sequence. The serines at 400 and 413 were known phosphorylation sites of Akt and the serine 443 is not a known phosphorylation site, but was recognized as a putative
CAMKII site (Kane et al., 2002; Scansite CAMKII data not shown). The lung mutation retained serines 400 and 413 but the thyroid mutation did not, and both mutations lost serine 443. Interestingly, the lung mutation sequence acquired a putative PDZ domain, a motif utilized in protein-protein interaction and localization of molecular signaling complexes (Harris and Lim, 2001). 49
Figure 3. Scansite analysis of the wildtype and mutant MAP3K8 amino acid sequences. The illustrations aligned from top to bottom are wildtype MAP3K8, lung mutant MAP3K8, and thyroid mutant MAP3K8, respectively. The sequences were analyzed under medium stringency. The program predicts the protein kinase domain from amino acids 137-389 in all sequences and three predicted serine phosphorylation sites in the carboxy terminus. Serine 443 lies within a phosphorylation motif that is lost in both mutants, while the lung mutant retains the serines 400 and 413 and acquires a PDZ domain.
To determine whether the mutation occurred in the original lung adenocarcinoma
DNA, we designed a MAP3K8-specific sense primer upstream of the mutation site
(5ACF) and a mutation-specific anti-sense primer downstream of the recombination site
(MUT-UX5). This primer combination would amplify only the MAP3K8/ chromosome 9 mutation. Genomic DNA from the lung adenocarcinoma (L-41), an immortalized lung cell line (9HTE), and several lung cancer cell lines were amplified with this primer combination. After amplification and DNA sequencing, L-41 was the only DNA to 50 amplify a product, confirming the mutation occurred in the primary lung tumor DNA
(Figure 4).
a.
b. Figure 4. The MAP3K8 mutation exists in the primary lung adenocarcinoma L-41. a) DNA isolated from L-41 and several cell lines was amplified with a MAP3K8 specific primer and a mutation specific primer. Only L-41 amplified a product demonstrating that the tumor contained the mutation. b) To confirm the presence of the mutation, 9HTE DNA (left) was sequenced with the sense primer and compared to the product amplified in L-41. Arrows indicate the mutation site.
In order to confirm the transforming potential of the mutated MAP3K8 gene, cDNAs of the wildtype and mutated gene were cloned into the eukaryotic expression vector pRc/CMV, transfected into NIH3T3 cells, and injected subcutaneously into nude mice. Transfections of the wildtype gene showed a moderate level of activity inducing tumors in 3 of 8 mice. The mutated MAP3K8 strongly induced tumors with a shorter latency and a higher frequency than the wildtype gene (Table 1). 51
Table 1. MAP3K8 induced tumor formation in nude mice. The empty pRc/CMV expression vector was transfected into NIH3T3 cells along with the pRc/CMV vector containing the MAP3K8 wildtype cDNA sequence (pRc/WT) and the MAP3K8 mutant cDNA sequence (pRc/MUT). The cells were injected subcutaneously into nude mice and tumor formation was assessed.
MAP3K8 mutations were a rare occurrence in lung cancer
Since previously characterized mutations of the MAP3K8 gene occurred in the
3’end of the open reading frame, we examined whether this was a common occurrence in lung cancers. Alterations of the 3’ end of the gene make standard RT-PCR impossible since one end of the sequence is unknown, but the 3’RACE technique overcomes this obstacle. cDNA was prepared from 22 of the lung cancer cell lines for the RACE experiment and the immortalized 9HTE cell line was used as a positive control.
Amplification was performed with a MAP3K8 specific primer (5ACF) designed to anneal prior to any of the known mutation sites and the 3’ primer supplied with the kit. The amplified product from all the cell lines tested was consistent with the expected molecular size of the product, approximately 1500 bases (Figure 5). 52
Figure 5. 3’RACE analysis of the MAP3K8 gene in lung cancer cell lines. RNAs were reverse transcribed from a variety of lung cancer cell lines with the SMART RACE kit (BD Biosciences Clontech, Palo Alto, CA). A primer was designed to anneal to the MAP3K8 Exon 7. Molecular weight markers are noted on the left. Lane 1 contains the immortalized lung cell line 9HTE as a positive control. None of the cell lines amplified a product that differed from the 9HTE wildtype gene.
To identify additional mutations occurring within the open reading frame of
MAP3K8, we designed six sets of primers for the coding region of the gene to amplify overlapping fragments approximately 300 bases in length (Table 2). The PCR fragments were denatured and run on SSCP polyacrylamide sequencing gels. Only one polymorphism, from cell line SK-LU-1, was identified in the 40 cell lines examined
(Figure 6b). DNA sequencing revealed this was an A/T heterozygous polymorphism residing in exon 7 of the MAP3K8 that did not alter the amino acid sequence of the protein (Figures 6c and 6d).
Table 2. Primer pairs for MAP3K8 PCR-SSCP. Primer pairs were designed in overlapping fragments to amplify the entire open reading frame of MAP3K8. The primer pairs and the size of their amplicon are listed in order from top to bottom. 53
a.
b. 54
c.
d.
Figure 6. SSCP analysis of the open reading frame of MAP3K8. Primers amplified overlapping fragments of the MAP3K8 gene in forty lung cancer cell lines. a) Amplification with MAP3K8-4AC-F and MAP3K8-4AC-R primers. Several of the cell lines did not amplify product suggesting the gene is down-regulated or absent in these cells. b) SSCP analysis of the MAP3K8-4AC-F/4AC-R product reveals a heterozygous polymorphism in the SK-LU-1 cell line. c) Sequence analysis of the SK-LU-1 sense (top) and anti-sense sequence (bottom). Arrows denote the polymorphism. d) Alignment of the wildtype sequence (Top) and the polymorphic sequence (Bottom) reveals no change in the amino acid sequence of the SK-LU-1 MAP3K8 protein. The polymorphic nucleotide is underlined and the predicted amino acids are centered below the codon.
Discussion
Several groups have independently identified the MAP3K8 gene as a transforming oncogene (Miyoshi et al., 1991; Patriotis et al., 1993; Chan et al., 1993). Here we identify an activating mutation of human MAP3K8 occurring in a primary human tumor and support the findings that over-expression of the wildtype gene from a strong promoter induces transforming potential in nude mouse tumorigenicity assays, but alterations of the carboxy terminus of MAP3K8 increase this potential a much greater degree. 55
We performed the NIH3T3 nude mouse tumorigenicity assay to identify transforming oncogenes from tumor DNA (Reynolds et al., 1991). DNA from a lung adenocarcinoma, L-41, was transfected into NIH3T3 cells and the cells were subcutaneously injected into nude mice. Human Alu probes confirmed the presence of human DNA in the developing tumors and hybridization of cloned DNA to a zoo blot suggested that the human DNA sequence contained exonic sequences conserved across primate species. The zoo blot positive clone was used to screen various cDNA libraries from the mouse tumors and identified MAP3K8 as the transforming gene. The MAP3K8 cDNA was sequenced to reveal an alteration at the 3’end of the open reading frame. To confirm that the mutated MAP3K8 induced the tumors, the cDNA was cloned into the pRc/CMV eukaryotic expression vector, transfected into NIH3T3 cells, and subcutaneously injected into nude mice. Over-expression of the wildtype MAP3K8 in
NIH3T3 cells demonstrated a moderate level of tumorigenicity causing tumor formation in 3 of 8 mice between 46 to 82 days post-injection. The mutated MAP3K8 gene demonstrated a much higher transforming ability, inducing tumors in 21 of 24 of the mice within 31 days post-injection. This data is consistent with other groups demonstrating a higher transforming potential in mutated MAP3K8 (Aoki et al., 1993; Ceci et al., 1997).
We performed a BLAST analysis of the lung mutation sequence and identified the mutation site as a region involving a genetic rearrangement or recombination with DNA from human chromosome 9. Compared to the wildtype protein of 467 amino acids, this alteration resulted in a shortened protein of only 429 amino acids and a different carboxy terminus. The mutation occurred in the penultimate exon of the MAP3K8 mRNA producing a novel 3’end and an altered carboxy terminus. Interestingly, the MAP3K8 56 mutation isolated from the thyroid tumor also demonstrated the mutation in the penultimate exon, but at a site upstream from the mutation we report (Miyoshi et al.,
1991). The Rat Tpl-2 mutation results from provirus insertion into the last intron, again resulting in an altered 3’end and changing the carboxy-terminus (Patriotis et al., 1993).
While all three mutations are different, all confer transformation potential to the gene through an alteration in the carboxy-terminus.
Activation of the MAP kinase pathway through Erk elicits the transformed phenotype in NIH3T3 cells (Janulis et al., 1999). In co-transfection experiments, MAP kinase cascade activation by oncogenic MAP3K8 was negatively regulated through physical association with the wildtype carboxy terminus (Ceci et al., 1997). Thus, it is probable that the mutated MAP3K8-induced transformation of NIH3T3 cells is through increased activation of the MAP Kinase pathway and the downstream protein Erk. The carboxy terminus of MAP3K8 is also required for binding to the p50 precursor NF-kB1
(Belich et al., 1999). Likewise, serines 400 and 413 in MAP3K8 are phosphorylation sites for Akt induced nuclear translocation of NF-kB (Kane et al., 2002). Interestingly, all three mutant forms lose serine 443, a potential CAMKII phosphorylation site according to the scansite program, but to date no functional relationship has been reported between
MAP3K8 and CAMKII. Thus, carboxy terminal alterations of MAP3K8 may affect NF- kB signaling pathways in addition to the classic MAP kinase pathways.
The MAP3K8 oncogene was first isolated by a procedure to transform SHOK cells with human thyroid tumor DNA (Miyoshi et al., 1991). However, the authors were unable to detect the mutation in the DNA from the original primary tumor, suggesting the mutation occurred during the transfection procedure. In order to confirm the mutation in 57 adenocarcinoma L-41 was not an experimental artifact, we designed primers to amplify the MAP3K8 mutation specifically. Through standard PCR with a wildtype MAP3K8 and a mutated MAP3K8-specific primer pair, the DNA from L-41 amplified a product.
The immortalized lung cell line 9HTE and other lung cancer cell lines did not amplify a product, demonstrating that the mutation occurred in the original primary tumor DNA and was not an artifact of the transfection procedure.
Next, we wanted to determine if 3’ alterations of MAP3K8 are a common occurrence in human lung cancer. 3’RACE was performed to identify major genetic alterations that would occur in the 3’end of the open reading frame. 3’ RACE adds a known sequence onto every mRNA reverse transcribed making amplification of unknown sequences possible. Of the cell lines we examined, none demonstrated additional alterations.
Unlike the lung and thyroid mutations, the MAP3K8 mutation found in rats occurs from MMLV provirus insertion into the last intron of the gene. Comparison of this intron sequence in humans and rats displays little homology even though the open reading frame of the gene is highly conserved (data not shown). Therefore, a similar method of proviral integration might not activate human MAP3K8.
The open reading frame of MAP3K8 was examined by overlapping fragments of
PCR-SSCP to identify point mutations. One polymorphism was identified in lung cancer cell line SK-LU-1 as an A/T heterozygous polymorphism in exon 7 of the MAP3K8 transcript. However, the polymorphism did not result in a change of the amino acid sequence. The SSCP data taken together with the 3’RACE data suggest that activating mutations of the MAP3K8 transcript are a rare event in lung cancer. 58
Certain genes display high frequencies of mutation in cancer. Ras mutation is a common occurrence in lung cancer as are mutations in the PI3 and Akt kinases in other cancers (Sekido et al., 1998, Blume-Jensen et al., 2001). Mutation of the Ras gene results in an activated protein product that stimulates the MAP kinase pathway. Constitutive activation and increased mRNA expression for many of the MAP kinases including Raf-1,
Mek, and Tak, are also tumorigenic, but, like MAP3K8, these genes are rarely mutated in lung cancer (Miwa et al., 1994; Bansal et al., 1997; Kondo et al., 1998). While these data support a role for MAP3K8 activity in lung tumorigenesis, the mutation identified in this report probably occurred through a rare genetic rearrangement during the cellular progression to cancer. Moreover, it is interesting that the mutations in the thyroid and lung tumors both occurred in exon 8, raising speculation as to whether this sequence is susceptible to recombination.
While additional mutations of MAP3K8 were not found, the PCR amplifications of the lung cancer cell line cDNA indicated that altered levels of expression may exist in lung tumors (data not shown). Such alterations of MAP3K8 mRNA transcription levels have been significantly associated with human breast cancer and overexpression of the wildtype gene displays mild transforming potential (Sourvinos et al., 1999; Chan et al.,
1993). Thus, it is possible that while mutational activation is a rare event, aberrant transcriptional regulation or gene amplification of MAP3K8 may be a common alteration occurring in lung cells and contribute to tumorigenic progression. 59
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Janulis M, Silberman S, Ambegaokar A, Gutkind J, and Schultz R. (1999). Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts. J Biol Chem, 274(2), 801-813.
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Chapter 5
Manuscript 2
Aberrant expression of the MAP3K8 proto-oncogene in lung cancer cell lines and the effect of MAP3K8 activity in lung cells 63
Title
Aberrant expression of the MAP3K8 proto-oncogene in lung cancer cell lines and the effect of MAP3K8 activity in lung cells
Authors: Adam Michael Clark1, Marshall Anderson2, and Jonathan S. Wiest1.
1. Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer
Research, National Cancer Institute, Bethesda, MD.
2. Department of Environmental Health, College of Medicine, University of
Cincinnati, Cincinnati, OH.
Acknowledgments: We would like to thank Mark Miller at the DNA Sequencing Core at the National Institutes of Health for his help with the Scansite program and primer extension analysis. 64
Abstract
MAP3K8 induces cellular transformation and tumorigenesis by transcriptional over-expression and mutation. While mutation of the gene appears to be a rare event in humans, altered MAP3K8 expression is significantly associated with several tumor types.
Here, we investigated MAP3K8 expression in lung cancer cell lines to determine if transcriptional aberrations are significantly associated with lung cancer. Primer extension,
5’RACE, and PCR-SSCP analyzed the MAP3K8 promoter; Realtime PCR and Western blot characterized transcriptional and translational expression; and FACS analysis examined the biological effects of increased expression in an immortalized lung cell line.
We report the promoter sequence as a GC rich, TATA-less promoter containing at least six transcription initiation sites. Realtime PCR demonstrated an increased expression of
MAP3K8 mRNA in non-small cell lung cancer cell lines but decreased expression in the majority of small cell lung cancer cell lines. Unexpectedly, stable transfection of either a wildtype or mutant MAP3K8 into a non-transformed lung cell line slowed proliferation in a non-apoptotic manner, but only the mutant gene arrested cells during G0/G1. These findings demonstrate that altered MAP3K8 expression is associated with tumorigenic lung cells, but over-expression in non-transformed cells suppresses growth. 65
Introduction
Lung cancer is the leading cause of cancer related deaths in the US and is divided into two major categories, non-small cell lung cancer (NSCLC) and small cell lung cancer
(SCLC) that differ at both a histological, as well as, a molecular level. Oncogenic alterations including p53 mutation, Ras activation, Bcl-2 up-regulation, Myc amplification, and loss of tumor suppressors p16 and Rb frequently occur during lung cancer development and progression (Salgia and Skarin, 1998). These genes participate in integral pathways affecting cellular growth, differentiation, survival, and apoptosis. The progressive accumulation of complementing tumorigenic alterations advances neoplastic development and illustrates the characteristic multi-step process of cellular initiation and promotion.
Alterations of growth permissive and suppressive pathways are hallmark characteristics of all cancers. K-Ras is a frequently mutated gene in lung adenocarcinoma, a sub-class of NSCLC, but rarely found mutated in SCLC (Sekido et al., 1998). Ras mutations constitutively activate the protein, stimulating downstream Raf and the MAP kinase pathway, a pathway conferring a growth advantage to NSCLC cells (Brognard and
Dennis, 2002). In contrast, the forced activation of the Ras/Raf/MAP kinase pathway in
SCLC cells induces growth inhibition and decreased tumorigenicity (Ravi et al.,
1998;Ravi et al., 1999). This discrepancy exists because the molecular contexts of the tumor cell types differ. MAP kinase activity complements frequently targeted alterations in NSCLC cells, such as loss of the tumor suppressor p16, to stimulate growth and 66 transformation. In contrast, inappropriate growth signals recognized and received by the molecular machinery within SCLC cells activate “dormant tumor suppressor pathways,” such as the p16 or p27 pathways, to slow growth and stop cellular transformation
(Weintraub, 1999). Dormant tumor suppressor pathways are growth inhibitive pathways retained by the cell but are functionally inactive. Recognition of inappropriate growth signals, such as Ras, reactivates the pathways and induces growth suppressive functions.
This indicates that the various types of lung cancers regulate genes differently in order to allow growth permissive versus suppressive functions.
MAP3K8 (also known as Cot and Tpl-2) has been repeatedly identified as a transforming protooncogene possessing similar cellular transforming activity to Ras and
Raf. MAP3K8 is a serine/threonine kinase that is a component in multiple signaling pathways including MAP and SAP kinase cascades, NFAT activation, degradation and nuclear translocation of NF-kB, and Caspase-9 induced apoptosis (Chariello et al., 2000;
Tsatsanis et al., 1998; Belich et al., 1999; Patriotis et al., 2001). Both mutation and over- expression of wildtype MAP3K8 are associated with cellular transformation and tumorigenesis, but mutation of the gene appears to be a rare event in humans (Miyoshi et al., 1991; Chan et al., 1993; Patriotis et al., 1993; Ceci et al., 1997; Eliopoulos et al.,
2002; Clark et al, submitted 2003). Increased expression of the MAP3K8 transcript was also a characteristic alteration in the early stages of breast cancer (Sourvinos et al., 1999).
On the contrary, the forced induction of MAP3K8 activity in primary mouse embryonic fibroblast (MEF) cells and the non-transformed fibroblast cell lines, HEK293 and REF52, resulted in slowed proliferation and activation the apoptotic pathway, respectively (Lund et al, 2002; Patriotis et al., 2001). These opposing activities demonstrate that in certain 67 cellular environments MAP3K8 activates tumor suppressor functions, while in others the protein activates its well-characterized oncogenic functions.
Previous studies have shown that increased transcriptional expression, increased protein half-life, and increased kinase activity of MAP3K8 all contribute to tumorigenic transformation. Transfected wildtype MAP3K8 under a strong promoter is sufficient to transform NIH3T3 cells and induce activation of downstream MAP3K8-specific pathways in the absence of stimulating conditions indicating that mRNA transcription is an important regulatory check point (Chan et al., 1993; Tsatsanis et al., 1998; Hagemann et al., 1999). In addition, carboxy-terminal truncated MAP3K8 displayed increased kinase activity and an increased half-life from resistance to proteolytic degradation contributing to its roles in cellular activation and proliferation (Gandara et al., 2003). However, it has been demonstrated that post-translational factors such as NF-kB1 binding negatively regulate MAP3K8 activity, but also increases protein stability by preventing degradation
(Waterfield et al., 2003). This indicates that multiple checkpoints exist regulating
MAP3K8 activity and abundance. Despite these discoveries, little is known about the relationships existing between MAP3K8 transcriptional expression, translation, and the subsequent influence on cellular growth and transformation.
In order to understand the role MAP3K8 plays in lung tumorigenesis we examined the expression characteristics of the gene in multiple lung cancer cell lines. We found the published MAP3K8 promoter was misreported and that the reported sequence actually maps to human chromosome 9p23, whereas the MAP3K8 gene is located at chromosome
10p11.2 (Sanchez-Gongora et al., 2000). We identify and characterize the MAP3K8 promoter as a GC rich, TATA-less promoter initiating transcription at multiple start sites. 68
In addition, we show increased MAP3K8 expression in a large fraction of NSCLC cell lines, but decreased expression in the majority of SCLC cell lines. However, stable transfection and expression of MAP3K8 of a mutant and a wildtype MAP3K8 in non- transformed lung cells demonstrated a decreased cellular proliferation rate without a change in the rate of apoptosis. These data suggest that while alterations of MAP3K8 expression are significantly associated with lung tumorigenesis, over-expression of the gene itself is not sufficient to induce a proliferative effect on non-transformed cell growth.
Materials and Methods.
5’RACE: 5’RACE was performed with the SMART RACE kit (BD Biosciences Clontech,
Palo Alto, CA) and the RACE reaction mixture was prepared according to the manufacturers instructions. The antisense primer sequence annealed to the first coding exon of the MAP3K8 transcript and had the following sequence. 5’-
CCTCCACAGTTCCATATCTG-3’. Thirty-five cycles of PCR was performed with the following parameters: 94 degrees C for 30 seconds, 65 degrees C for 30 seconds and 68 degrees C for 120 seconds. The products were analyzed on a 1% agarose gel, purified, and sequenced.
Fluorescent Labeled Primer Extension: Total RNA was isolated from the cell lines.
Twenty-five ug of RNA was incubated at 65 degrees C for 10 minutes and mixed with 30 units Omniscript Reverse Transcriptase (Qiagen, Valencia, CA), 40 ng/uL FAM labeled primer (5’-TGACTCACGGCGTCTGAGCCTGCCCA), 0.5 mM dNTP, and 10X Buffer
RT to complete the final reaction mixture. The mixture was then incubated at 37 degrees 69
C for 60 minutes and the reverse transcriptase was deactivated at 93 degrees C for 5 minutes. Ten units of RNase was added to the mixture and incubated at 37 degrees C for
60 minutes. The cDNA was purified with the QIAquick PCR purification column
(Qiagen Inc., Valencia, CA), diluted in deionized water, run on an ABI3100 DNA
Sequencer (Applied Biosystems, Foster City, CA), and analyzed through the Scanalyze program (See acknowledgements).
Promoter Analysis: Genomic DNA was isolated from 39 lung cancer cell lines and three
PCR fragments were amplified with the following primer pairs: Set A (5’-
CTCTGTCCCCACCCCAGAGCGTG-3’/GACTAGGGAGGAGCAGAGCGG-3’);Set B
(5’-AGCTCACGTCCCGCGCTCCTCCCT-3’/5’-
ACCGCCTCCCCGACTTTTCCATTTC-3’); Set C (5’-
GCAACCGGGCAGTCTCTTTCTGTTTAC-3’/5’-
CAGAAGCAGGAACCAGGGCGCC-3’). The fragments were amplified according to standard PCR conditions, denatured, and run on a 0.5X MDE sequencing gel
(BioWhittaker Molecular Applications, Rockland, ME) with and without 5% glycerol.
The DNA products were visualized through silver staining techniques (Cat #Q4132,
Promega, Madison, WI) according to the manufacturer’s instructions. Samples displaying altered migration patterns were sequenced for confirmation of altered nucleotide sequence with the BigDye Terminator Reaction Mix (Applied Biosystems, Foster City, CA) fluorescent sequencing kit. 70
Realtime PCR: 1 ug of total RNA was reverse transcribed into cDNA. Primers were designed spanning exon 5 to exon 7 (5’-CTACACTCAAAGAAAGTGATCCATC-3’/ 5’-
GATGACCTCTGGGCTCATGTAAA-3’) of the MAP3K8 gene and normalized to the
GAPDH expression levels. The transcription levels were measured and quantified on the
ABI7900 (Applied Biosystems, Foster City, CA) by Sybr green incorporation into the double stranded DNA. The samples were amplified in duplicate for each primer set. The
MAP3K8 Realtime threshold levels (Ct value) were calculated and normalized to the
GAPDH amplification levels by subtracting the GAPDH Ct value from the MAP3K8 Ct value. The relative difference in expression was determined by calculating the difference between the normalized lung cancer cell expression level and the normalized Normal
Human Bronchial Epithelial (NHBE) cell level. Each cycle difference in normalized Ct level amplification corresponded to a two-fold difference in expression. A four-fold difference in expression was considered to be significantly different.
Western Blot: Whole cell protein lysate (60 ug) was loaded onto a 10% Bis-Tris NuPage gel (Invitrogen, Carlsbad, CA) and run overnight at 35V. The protein was transferred onto a nitrocellulose membrane (Invitrogen, Carlsbad, CA), blocked with 5% Blotto
(Santa Cruz Biotechnology, Santa Cruz, CA), and labeled with 1:500 dilution of anti-
MAP3K8 (Cat #H212, Santa Cruz Biotechnology, Santa Cruz, CA). The westerns were developed under normal conditions after binding with a 1:10,000 HRP conjugated secondary (Cat#NA934V, Amersham Biosciences, Piscataway, NJ). Values were determined through densitometry levels normalized on actin levels (1:5000 mouse anti- 71 actin, Cat#5441, Sigma-Aldrich, Milwaukee, WI. and 1:20,000 sheep anti-mouse,
Cat#NA931V, Amersham Biosciences, Piscataway, NJ).
Cell Transfection and Growth: Cells were transfected with GenePORTER 2 transfection reagent (Gene Therapy Systems, San Diego, CA) with pRc/CMV (Invitrogen, Carlsbad,
CA) expression vectors and maintained in selective media with 400 ug/mL G418 (Sigma-
Aldrich, Milwaukee, WI.) in DMEM with 10% FBS (Gibco, Carlsbad, CA). The pRc/WT plasmid contains the wildtype MAP3K8 cDNA transcript and pRc/MUT contains a transforming mutant MAP3K8 cDNA under control of the CMV promoter. For growth curve analysis, cells were plated in triplicate in 3.5 cm dishes (1000 cells per plate done in triplicate) and grown in DMEM + 10%FBS + 400ng/mL G418 for a period of 16 days.
Cells were trypsinized and counted in a counting chamber on the selected days. The cell number was graphed over time with standard error for each day counted.
FACS Analysis: Cell cycle analysis was measured with propidium iodide nuclear labeling.
In brief, the cells were fixed in 70% ethanol, treated with RNase, and labeled with propidium iodide. The cells were analyzed in the FACSCalibur FACS machine (BD
Biosciences, Franklin Lakes, NJ) in triplicate and average values with standard deviations were taken for each of the populations. Apoptotic cells were measured through TUNEL assay analysis with the APO-BrdU TUNEL Assay kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. 72
Homogenous Caspase Assay: Cells were collected and analyzed for caspase activity using the Homogenous Caspase Assay kit (Roche, Basal, Switzerland) according to the manufacturer’s instructions. Camptothecin treated U937 cells were used as a positive control for caspase activity. Caspase activity of the transfected cell lines was measured with 4000 cells per well in sets of 8 wells per cell line and the standard error was calculated.
Computer Analysis: BLAST searches to identify the promoter location were performed on the NCBI BLAST search engine at http://www.ncbi.nlm.nih.gov/BLAST/ and the
Ensembl search engine at http://www.ensembl.org. The transcription factor binding site motifs were identified through the MatInspector program at http://www.genomatix.de/cgi- bin/matinspector/matinspector.pl/.
Statistical Analysis of the Growth Curve: In order to establish statistical significance for the cellular growth, the mean and standard deviations were calculated and analyzed as independent populations using the paired t-test with a P=0.05.
Results
Identification of the MAP3K8 promoter
Our initial examination of MAP3K8 expression began with an analysis of the promoter. BLAST analysis (data not shown) using the published promoter sequence suggested it was incorrectly identified and characterized (Sanchez-Gongora et al., 2000).
The published promoter aligned to a portion of clone RP11-141L16 that mapped to a 73 region on human chromosome 9p23. The NCBI BLAST analysis identified the MAP3K8 gene location to human chromosome 10p11.2(identified as a partial sequence in Genbank accession number AF133211), a location that was confirmed by analysis with the Ensembl search engine. To identify the correct promoter sequence, we designed an antisense primer to anneal to the first coding exon of the MAP3K8 transcript, 245 nucleotides from the translation initiation site, and amplified towards the 5’ end of the gene in a 5’RACE reaction. Two products amplified from the CHAGO-K-1 and the H460 cell lines of approximately 509 and 740 base pairs (Figure 1a). The products were isolated, purified from the gel and sequenced. Sequence analysis revealed that the PCR products aligned to human clone RP11-449I17 that extends along chromosome 10p11.2 and was contiguous with the MAP3K8 gene. The smaller of the two products resulted from alternative splicing of the second exon of MAP3K8 (Figure 1b) as previously reported (Sanchez-
Gongora et al., 2000).
a. 74
b.
Figure 1. 5’ RACE identifies the putative MAP3K8 promoter region and an alternatively spliced exon. a) A MAP3K8 antisense specific primer annealed to the beginning of the MAP3K8 open reading frame in exon 3 and amplified through the 5’ end. Two products amplified in both CHAGO-K-1 and H460 cell lines that result from an alternative spliced non-coding second exon. b) Illustration of the MAP3K8 intron/exon splicing from chromosome 10p11.2. MAP3K8 is composed of 9 exons with exon 2 is alternatively spliced. The boxes illustrate each exon and the relative position within the genome. Shaded boxes represent the coding regions of the gene and the empty boxes represent the non-translated regions.
Sequencing through the entire amplicon provided a region to examine for the promoter analysis. Based on the 5’RACE sequencing data, a FAM labeled primer was designed to anneal to the first exon of the MAP3K8 transcript near the end of the 5’RACE amplicon. Total RNA from one immortalized lung cell line (9THE) and five lung cancer cell lines (A549, H322, H345, H441, H460) was reverse transcribed and analyzed by fluorescent labeled primer extension. The results from the primer extension for the large cell lung cancer cell line H460 and the adenocarcinoma cell line A549 are illustrated in
Figure 2a and are labeled representatives of the six cell lines examined. All six cells lines are shown in Figure 2b, but are scaled down and the peaks are not labeled.
In the figures, each blue peak corresponded to a reverse transcription product resulting from the primer extension reaction. The red peaks were the TAMRA 500 molecular weight standards run in each sample lane as an internal standard to determine 75 the nucleotide length of the products. At least six transcription initiation sites were identified (Table 1). PEAK239 was the only initiation site identified in all the cell lines examined, but the PEAK101 and PEAK144 appeared in multiple cell lines (peak numbers correspond to the number of nucleotides attached to the FAM label, thus the PEAK239 is
239 nucleotides long). The additional peaks listed were unique to the cell lines examined.
A region of 320 base pairs encompassing all the transcription initiation sites was identified as the putative promoter. The sequence was analyzed with the MatInspector program and found to be GC rich, TATA-less promoter that contained multiple transcription factor binding site motifs (Figure 3). 76
a. 77
b.
Figure 2. Fluorescent labeled primer extension of the MAP3K8 RNA. Fluorescent labeled primer extension of cell line RNA reveals multiple transcription initiation sites in the MAP3K8 promoter. FAM labeled primer extension products (blue peaks) were reversed transcribed from cell line RNA and run along with TAMRA 500 molecular size standards (red peaks). a) The cell lines H460 (top) and A549 (bottom) are representative examples. Each blue peak corresponds to a transcription initiation site for the gene. The number denoted above the peak is the number of nucleotides extending from the FAM tag to the initiating nucleotide. b) The six cell lines examined. The name of the cell line is in the upper right-hand corner of each sample. All of the cell lines were examined and the peaks listed in Table 1. 78
Cell Cell PEAK12 PEAK12 PEAK14 PEAK2 PEAK2 PEAK2 Line Type PEAK101 4 9 4 16 35 39 9HTE Normal X ! ! X ! ! X A549 Adeno X ! ! X X ! X H441 Adeno X ! ! X ! X X H322 Adeno ! ! ! ! ! ! X H460 Large X X X ! ! ! X H345 Small X ! ! X ! ! X
Table 1. Transcription initiation sites. Six cells lines were analyzed by primer extension (Figure 2) and expression of the transcription initiation sites for each of the cell lines is marked with an X for positive expression of the site.
Figure 3. Analysis of the MAP3K8 promoter region. Nucleotide sequence of the MAP3K8 promoter. The polymorphic nucleotides are shown with their corresponding nucleotide above in parentheses. The MAP3K8 mRNA start sites are shown in bold with the PEAK values listed above. Transcription factor binding site motifs are underlined and identified by the putative transcription factor below. 79
The MAP3K8 promoter contained two polymorphisms
In order to identify mutations/polymorphisms contained within the promoter, primers named Primer Set A, B, and C were designed for PCR-SSCP (Single Strand
Conformational Polymorphism) analysis to produce overlapping fragments 310, 240, and
224 base pairs long. SSCP identified two products with altered migration patterns (Figure
4) and were confirmed to be polymorphisms through DNA sequencing analysis (data not shown). SSCP analysis with Primer Set B identified a G/C polymorphism residing in the core-binding domain of the Wilms Tumor Suppressor (WT1) transcription factor binding site motif. Of the 39 lung cancer cell lines examined, nine cell lines were homozygous for
G, sixteen cell lines were homozygous for C, and fourteen cell lines were heterozygous at this site (Table 2). Primer set C identified a G/A polymorphism found downstream of the
G/C polymorphism. Only cell lines H69 and H322 contained this polymorphism and both were heterozygous. All other cell lines were homozygous for the guanine. The G/A polymorphism did not reside in any identified transcription factor core binding domain motif. In addition, no correlation was found between the genotype of either polymorphism and lung cancer histology. 80
Figure 4. PCR-SSCP of the MAP3K8 promoter. Lung cancer cell lines were analyzed with three different primer sets to identify polymorphisms within the MAP3K8 promoter. Two polymorphisms were identified, represented in the figures above. (Left) A G/C polymorphism was identified from amplification with Primer Set B. CHAGO-K-1 was characteristic of the guanine polymorphism, whereas the CALU1 cell line is characteristic of the cytosine polymorphism. (Right) A G/A polymorphism was identified from amplification with Primer Set C. Cell lines H69 and H322 were heterozygous for the adenine nucleotide. All other cell lines were homozygous for guanine. P3K8 RNA 81
Cell Line Tumor Type Primer Set B Primer Set C 9HTE NSCLC C G H125 NSCLC G/C G H157 NSCLC C G H324 NSCLC G G H441 NSCLC C G H596 NSCLC C G COLO699 NSCLC C G H726 NSCLC G/C G H727 NSCLC G/C G SKLU-1 NSCLC C G A549 NSCLC C G H2122 NSCLC G G H792 NSCLC G/C G H460 NSCLC G/C G H661 NSCLC G G H1334 NSCLC G G H720 NSCLC C G NE18 NSCLC G/C G H322 NSCLC G/C G/A H358 NSCLC C G CALU6 NSCLC G/C G CHAGOK-1 NSCLC G G CALU1 NSCLC C G CALU3 NSCLC C G UCLC11 NSCLC G G H1264 NSCLC C G SKMES-1 NSCLC G G SW900 NSCLC C G H69 SCLC G/C G/A H345 SCLC G G H372 SCLC C G H524 SCLC G G H735 SCLC C G H1062 SCLC G/C G H1607 SCLC G/C G H1341 SCLC G G H446 SCLC C G N417 SCLC G/C G SHP77 SCLC G G
Table 2. MAP3K8 promoter polymorphisms within lung cancer cell lines. Results of the PCR-SSCP analysis of various NSCLC and SCLC cell lines. A G/C polymorphism was identified within the Primer Set B amplification sequence and a G/A polymorphism was identified within the Primer Set C amplification sequence. Cell lines that were also analyzed by primer extension are labeled in boldface. 82
MAP3K8 transcription was differentially expressed in lung cancer cell lines and correlates with lung cancer histology
We examined MAP3K8 expression levels in lung cancer cell lines to determine if the levels were associated with tumorigenesis and cellular transformation as in other cell lines and tumor cells (Aoki et al., 1993;Chan et al., 1993;Sourvinos et al., 1999). Using
Realtime PCR we amplified a fragment of cDNA that spanned MAP3K8 exons 5-7 in seventeen NSCLC and eleven SCLC cell lines and compared the relative expression levels after normalization to GAPDH expression (Figure 5). Total RNA isolated from NHBE
(Normal Human Bronchial Epithelial) cells was used as the normal expression level and assigned a relative expression value of one. The immortalized bronchial epithelial cell line 9HTE was also amplified as a non-tumorigenic lung cell line for comparison. The reactions were performed in duplicate and expression values were characterized as fold differences from the NHBE values. Only values exhibiting a 2-cycle or greater PCR difference were considered to be significantly different in expression. The 2-cycle difference correlated to a 4-fold increase of relative expression in cells with increased expression of the gene or a 0.25 relative expression in cells with decreased expression of the gene. 83
a. 84
b.
c. 85
d.
Figure 5. Realtime PCR of the MAP3K8 cDNA. The four graphs represent the relative MAP3K8 expression among the various cell lines and are designated as increased (a) and decreased (b) expression in NSCLC cell lines and increased (c) and decreased (d) expression in SCLC cell lines. All values are based on the expression level of normal human bronchial epithelial cells (NHBE) and normalized by expression of GAPDH levels. The bar graphs depict the relative expression based on a NHBE value of 1. Therefore in the Increased Expression graphs a fold difference of 10 would represent 10 times the amount of MAP3K8 in the cell line as in the NHBE cells, whereas in the Decreased Expression graphs, a value of 0.1 would represent a 10-fold decrease in expression compared to the NHBE cells.
As expected, the non-transformed 9HTE cell line expressed MAP3K8 at values that were much less than a two-cycle difference from the NHBE cells and, thus, were not significantly different resulting in a 2.2-fold relative expression compared to NHBE. Of the NSCLC cell lines the general trend was demonstrated to be increased expression, but seven of seventeen had significantly increased MAP3K8 transcript levels and three additional cell lines showed a trend towards increased expression but were not significantly different (Figure 5a). Among these, CHAGO-K-1, A549, and CALU3 had increased expression levels greater than 50-fold over the NHBE cell expression levels.
Only two of the NSCLC cell lines (H720 and NE-18) had significantly lower expression 86 levels (Figure 5b), and both these had a relative expression less than 0.01 that of the
NHBE expression values (100 fold lower difference). Of the SCLC cell lines four of eleven had significantly higher expression levels (Figure 5c), six of eleven had significantly lower levels, and one cell line was not statistically different from NHBE cells
(Figure 5d).
Lack of concordance between MAP3K8 protein levels and Realtime PCR expression levels
We next examined if the MAP3K8 protein exhibited similar levels by Western blots as the mRNA levels by Realtime PCR in the cell lines. The MAP3K8 protein is translated into two isoforms from alternative transcription start sites in the first coding exon. Whole cell protein lysates were collected from the 29 cell lines and analyzed by
Western blot with a MAP3K8 specific antibody (Figure 6). As with the Realtime PCR, a variety of characteristics were observed for the MAP3K8 protein in the cell lines. These ranged from no detectable expression, to high expression, as well as variations between the a (larger) and b (smaller) isoforms. The Western blot data demonstrated that the mRNA and protein levels did not correlate. The NSCLC cell lines such as CHAGO-K-
1and A549 significantly up-regulated the mRNA greater than 50-fold over the NHBE cells, yet they did not show detectable levels of protein product. The cell line H460 was the only NSCLC cell lines that appeared to correlate the increased transcript with increased protein product. In contrast, the NSCLC cell lines NE-18 and H720 displayed high levels of protein product; yet transcribe very low levels of MAP3K8 mRNA. Among the SCLC cell lines, H69 and H748 both had reduced mRNA expression levels, but 87 display detectable protein products. With the exception of cell line H792, the SCLC cell lines that displayed increased expression of the transcript correlated this with increased
MAP3K8 protein products. Interestingly these cell lines also displayed both isoforms
(Table3). 88
Table 3 (above). Comparison of MAP3K8 expression data. The table compiles the Realtime PCR and western blot data. The relative mRNA expression is listed in the third column in ascending order, separated by the NSCLC and SCLC cell lines. The 9HTE immortalized cell line is also listed for comparison. The relative expression values from the western blots are listed next to the mRNA levels. An X in the last two columns indicates whether the cell lines expressed the a or b isoforms of the protein.
Figure 6. MAP3K8 Western blot analysis of the lung cancer cell lines. The cell lines analyzed by Realtime PCR were compared for MAP3K8 protein expression. There are two translation products, a (top band) and b (bottom band) of MAP3K8 resulting from alternative AUG start sites. The 9HTE immortalized lung cell line was used as a normal control for the blots. The expression levels varied among the cell lines examined, as did the expressed protein isoforms.
Stable transfection of MAP3K8 reduced 9HTE cell proliferation
We examined what effect MAP3K8 transfection and increased expression would have on the immortalized bronchial-epithelial cell line 9HTE. Plasmid constructs of
MAP3K8 in the pRc/CMV eukaryotic expression vector were generated and transfected into the 9HTE cells. Empty pRc/CMV vector (9HTE-CMV), wildtype MAP3K8 cDNA
(9HTE-WT), and oncogenic MAP3K8 cDNA mutant (9HTE-MUT) plasmids were stably 89 transfected into the immortalized 9HTE cell line, kept under selective pressure with the
G418 antibiotic, and subjected to analysis.
Figure 7. Stable transfection of the MAP3K8 wildtype and mutant gene into 9HTE cells. (Left) MAP3K8 cDNA was inserted into the pRc/CMV eukaryotic expression vector and transfected into 9HTE cells. Amplification with MAP3K8 exon specific primers confirmed stable transfection of the vector into the 9HTE genome, and cDNA amplification displayed increased levels of transcribed MAP3K8 compared to the empty vector controls. Amplification with mutation specific primers confirmed expression of the mutant transcript in the 9HTE-MUT cells. (Right) Western blot analysis demonstrates increased expression of the MAP3K8 wildtype and mutant protein in the transfected cell lines.
In order to confirm the stable transfection of the plasmids into the 9HTE genome, we analyzed the genomic DNA, RNA, and protein of the different transfected cell lines
(Figure 7). Exon-specific primers amplified the MAP3K8 cDNA sequence from the genomic DNA of the 9HTE-WT and 9HTE-MUT cells but not from the 9HTE-CMV cells confirming the plasmid constructs had stably integrated into the 9HTE genome. The
MAP3K8 transfected cells also amplified higher levels of MAP3K8 transcript from the 90 cDNA compared to the empty vector transfected cells confirming increased expression of the gene under the CMV promoter. In addition, the transfected cell line cDNAs were amplified with a MAP3K8 mutation specific primer to confirm transfection of the mutant cDNA. The 9HTE-MUT cells but not the 9HTE-CMV or 9HTE-WT cells produced the mutated transcript as demonstrated by PCR confirming transfection and expression of the mutant sequence (Figure 7). When analyzed by western blot, both the 9HTE-WT and
9HTE-MUT cells showed increased levels of both isoforms of the MAP3K8 protein product but the 9HTE-CMV cells did not, further confirming that the genes were not only transcribed but also translated (Figure 7).
Since transfection of wildtype and mutant MAP3K8 induces cell cycle progression in T cell lymphocytes but slows proliferation in MEF (Mouse Embryonic Fibroblast) cells, a growth curve was generated to analyze the effect of MAP3K8 in the lung cell line
9HTE (Velasco-Sampayo and Alemany, 2001; Lund, et al.,2002). The transfected cells were seeded in equal numbers, maintained in G418 selective medium, and counted over a
16-day period (Figure 8a). By day 8, a noticeable difference occurred in the proliferation rate of the transfected cells. Both wildtype and mutant MAP3K8 over-expressing cells demonstrated a reduced proliferation rate. By day 12 this difference became significant for the 9HTE-WT cell line (P=0.05) and by day sixteen the difference became significant for the 9HTE-MUT cell line (P=0.05). Over the course of 16 days the empty vector
9HTE-CMV cells grew from a population of 1000 cells to 140,000 cells. In comparison, the wildtype and mutant MAP3K8 transfected cells had nearly identical proliferation rates growing from 1000 cells to about 85,000 cells over the 16 day period. These values 91 correlate to a doubling time of 2.24 days for the 9HTE-CMV cells and a doubling time of
2.49 days for the 9HTE-WT and 9HTE-MUT cells.
a.
b.
Figure 8. Expression of MAP3K8 wildtype and mutant genes slows 9HTE proliferation. a) Growth curve of 9HTE transfected cell lines. 1000 cells were plated in triplicate and grown over a period of 16 days. The 9HTE empty vector cell line proliferated at a higher rate than both the wildtype and mutant transfected cells, which appeared to have identical 92 proliferation rates. b) Flow cytometry of the transfected populations demonstrates that the mutant MAP3K8 transfected cells were arrested in the G0/G1 phase of the cell cycle, but the empty vector and wildtype transfected cells did not display the G0/G1 cell cycle arrest.
Cell cycle profiles differed between wildtype and mutant MAP3K8 transfected cells without a change in apoptosis
Both arrested cell cycle progression and increased rate of cell death can decrease the cellular proliferation rate. The transfected cells lines were examined with FACS analysis to determine alterations within the cell cycle that might account for the decreased proliferation rates (Figure 8b). Interestingly, although 9HTE-CMV and 9HTE-WT had different growth rates, they displayed similar cell cycle characteristics. There was a slightly greater increase in the percentage of 9HTE-WT cells in the G0/G1 phase of the cell cycle compared to the 9HTE-CMV cells, but the S and G2/M phase cells showed no difference. However, the 9HTE-MUT cells had a dramatically increased population of the cells in the G0/G1 and S phases of the cell cycle with a decreased proportion of the cells in the G2/M phase. About 45% of the 9HTE-MUT cells were held in G0/G1 compared to
34% and 36% of the 9HTE-CMV and 9HTE-WT cells, respectively. But only 27% of the
9HTE-MUT cells were in G2/M, compared to 44% and 43% of the 9HTE-CMV and
9HTE-WT cells, respectively (Table 4).
CELL %G0/G1 %S %G2/M
9HTE-CMV 33.9 ± 0.52 22 .16 ± 0.48 43.97 ± 0.16
9HTE-WT 36.29 ± .09 20.94 ± 1.39 42.77 ± 1.23
9HTE-MUT 45.39 ± 0.71 27.57 ± 0.35 27.04 ± 1.08 93
Table 4. Cell cycle analysis of transfected 9HTE cells. Cells were transfected with the different vectors and subjected to FACS analysis. Each population was analyzed in triplicate. The numbers represent the percentages of cell in each phase of the cell cycle along with the standard deviation.
Since the 9HTE-WT cells did not display the same cell cycle profile as the 9HTE-
MUT cells but did display a similar proliferation rate, we examined the possibility that wildtype MAP3K8 slowed proliferation through induction of apoptosis. MAP3K8 can activate the Caspase-9/Caspase-3 apoptotic pathway (Patriotis et al., 2001). To test if this was occurring in the transfected cells, we analyzed the cells by the TUNEL assay with apoptosis specific markers that label fragmented DNA, a hallmark of apoptosis (Figure
9a). Triplicate samples of each transfected cell lines was labeled, sorted for expression of the apoptotic marker, and counted as a percentage of the total cells. Each of the transfected cell lines had populations of between 2-3% of the total number of cells undergoing apoptosis, but the percentage of 9HTE-WT and 9HTE-MUT cells undergoing apoptosis was not different from the 9HTE-CMV cells. 94
a.
b.
Figure 9. MAP3K8 does not induce apoptosis in 9HTE cells. a) TUNEL analysis of the empty vector, wildtype, and mutant transfected cells indicates that the number of cells undergoing apoptosis is not significantly different among the populations. The percentage of cells undergoing apoptosis along with the standard deviation is listed below the figure. b) The cells were analyzed through a homogenous caspase activity assay and measured by Relative Fluorescence Units (RFU). Lane 1 depicts the U937 camptothecin treated 95 positive control cells undergoing caspase induced apoptosis. There was no significant difference in caspase activity among the transfected cell lines examined. Eight samples of cells were analyzed and graphed along with the standard error.
To confirm that the cells did not have subtle differences in apoptotic rates that were not recognized by a TUNEL assay, we measured the relative caspase activity of the cells. The caspase assay measured the activity of the major caspases without selecting for one particular pathway (Figure 9b). U937 cells treated with camptothecin were used as a positive control for apoptotic induction. Each cell line was analyzed in sets of eight, but neither the 9HTE-WT or 9HTE-MUT cells showed any significant change in caspase activity relative to 9HTE-CMV cells.
Discussion
The purpose of this research was to analyze various MAP3K8 expression characteristics in lung cancer cell lines to determine if they were associated with and contributed to lung tumorigenesis. Here, we identified and characterized the MAP3K8 promoter as a TATA-less, polymorphic promoter with multiple transcription initiation sites. We demonstrated that the MAP3K8 gene was differentially regulated at both a transcriptional and translational level in various histologies of lung cancer cell lines indicating that altered expression was associated with lung tumorigenesis. We also showed that transfection of the gene in a non-transformed cell line unexpectedly slowed proliferation of the cells in a non-apoptotic manner. While the results demonstrated that altered expression of MAP3K8 occurs in lung cancer cell lines, the growth suppressive characteristics expressed by the non-transformed lung cells suggested that other 96 alterations are necessary to complement an oncogenic function of the gene in lung tumorigenesis.
The paradoxical relationship of the same gene regulating growth progression and suppression has been previously demonstrated in lung cancer cells. Activation of signaling proteins in the MAP kinase pathways influences cellular proliferation, tumorigenic progression, and chemotherapeutic resistance in human NSCLC (Brognard and Dennis, 2002). In contrast, activation of these same pathways in SCLC induces an opposite growth response resulting in slowed growth and decreased tumorigenicity (Ravi et al., 1998; Ravi et al., 1999). This is due to the purported “dormant tumor suppressor” pathways (Weintraub, 1999). These pathways are functional in the SCLC cells, but inactive. The SCLC cellular machinery recognizes inappropriate induction of growth signaling pathways, such as those induced by Ras of Raf activation. In turn, the cells activate tumor suppressor genes, such as p16 or p21, and arrest the growth of the cell to counteract the mitogenic activity (Weintraub, 1999). The differences between the molecular etiologies of non-small versus small cell lung cancers and the pathways involved with these different cellular contexts account for the different responses to abnormal MAP kinase activity.
The MAP3K8 gene has also demonstrated both oncogenic and tumor suppressive characteristics in different cell types suggesting that the gene may be a key regulator of these activities. Increased expression of the wildtype gene and mutation of the carboxy terminus of the protein are two means by which MAP3K8 confers transformation and tumorigenic potential to a cell (Ceci et al., 1997; Chan et al., 1993; Miyoshi et al., 1991).
On the other hand, MAP3K8 induces a G0/G1 arrest and participates in Caspase- 97
9/Caspase-3 induced apoptosis when over-expressed in several non-transformed cell lines
(Lund et al., 2002; Patriotis et al., 2001). The functional role of MAP3K8 in lung cells has not been determined, but we have shown that mutational activation of the gene is a rare event in lung cancer (Clark et al., 2003, manuscript in submission). However, as with the increased expression of the gene in breast cancers, alterations outside of mutation that regulate expression and activity may be important alterations in lung tumorigenesis.
We identified the MAP3K8 promoter through a combination of BLAST search analysis and 5’RACE in order to clarify the discrepancy between the literature and the databases. The 5’RACE analysis generated two transcripts in cell lines CHAGO-K-1 and
H460. The smaller of the two transcripts corresponded to a transcript with an alternatively spliced non-coding second exon that had been previously reported ((Sanchez-Gongora et al., 2000)). Sequence analysis confirmed that these products mapped to a region on chromosome 10p11.2 and were contiguous with the MAP3K8 gene (data not shown). The termination site of the 5’RACE products was located within a GC rich region that did not contain transcription start site elements such as a TATA or CAATT box suggesting that the MAP3K8 gene was generated from a TATA-less promoter.
To analyze for transcription initiation sites we developed a fluorescent-labeled primer extension procedure. Total RNA from several cell lines was reverse transcribed with fluorescent FAM labeled primers specific to MAP3K8 exon one. Primer extension revealed at least six different transcription initiation sites within the promoter, a common characteristic of GC-rich, TATA-less promoters (Smale, 1997). Only one initiation site, named PEAK239 was observed in all the cell lines examined. Sites such as PEAK101 and PEAK144 were found in multiple cell lines. The sample size was too small to 98 determine if these site differences were due to the tumor histology or had any significance in transcriptional regulation. However, it is interesting that the only large cell lung cancer cell line examined, H460, was also the only cell line to contain PEAK124 and PEAK 129, initiation sites that lie downstream the NF-kB binding site motif. Clusters of peaks approximately 40 and 60 bases from the fluorescent labeled primer were observed. These clusters were found in all the cell lines examined, but it is not clear if they were actual transcription initiation site clusters or existed as fluorescent-labeled artifacts from the primer extension reaction.
After identification of the promoter, DNA from various cell lines was examined by
PCR-SSCP to characterize polymorphisms in the MAP3K8 promoter. Two polymorphisms were identified in the region. A G/C polymorphism resided in the core binding domain of the Wilms Tumor Suppressor (WT1) transcription factor binding site motif. Both the guanine and the cytosine appeared to have a nearly balanced frequency, although the cytosine was slightly favored. This was not the case with the second polymorphism; a G/A polymorphism located further downstream that was heterozygous in two cell lines, H69 and H322, but homozygous for the guanine in the other 37 cell lines.
This second polymorphism does not reside in any known transcription factor binding site motif, suggesting that it may not have any functional effects on transcriptional regulation.
However, neither polymorphism was examined in normal DNA samples to ascertain if there is an unusual frequency in the lung cancer cell lines.
We analyzed MAP3K8 expression to determine if altered mRNA expression occurred in lung cancer as has been previously demonstrated in breast cancer (Sourvinos et al., 1999). NHBE cells were used as a normal control for MAP3K8 expression levels. 99
The 9HTE immortalized cell line was used for comparison. Realtime PCR quantified the relative expression levels of MAP3K8 in the various cell lines. The cells lines CHAGO-
K-1, A549, and CALU-3 had greater than 50-fold increased expression of MAP3K8 relative to the NHBE cells, suggesting that over-expression of MAP3K8 may be an important factor in NSCLC tumorigenesis, at least in a fraction of the cases. Among the
SCLC cell lines, over half significantly decreased MAP3K8 expression compared to
NHBE cells suggesting that alternative regulation of the gene may exist between the different lung cancer histologies. If the increased transcription ultimately resulted in increased MAP kinase activity, the alteration could be tumor promoting in NSCLC cells.
On the contrary, the decreased transcription and inactivation of these pathways in SCLC might prevent initiation of the “dormant tumor suppressor” pathways, thereby facilitating tumorigenic growth of these cells.
To complement the Realtime data, we analyzed the MAP3K8 protein by western blot in the different cell lines. Protein expression did not correlate with mRNA expression in many of the cell lines. In cell lines NE-18 and H720, both of which show extremely low levels of RNA, the protein levels were inversely proportional, displaying distinct bands in the western blot. Alternatively, cell lines with marked increased expression,
A549 and CHAGO-K-1, did not display detectable levels of protein. The western blot results suggested that multiple factors participate in protein translation and stability.
The MAP3K8 gene encodes two isoforms, a and b, translated from separate translation initiation sites. There was a great degree of variation among the translated isoform in the different cell lines. However, the only apparent consistency was that the
SCLC cell lines demonstrating detectable levels of protein appeared to produce both 100 isoforms. The RNA and protein data taken together indicate that the different histologies may process MAP3K8 differently at transcriptional, translational, and post-translational level.
It is unclear why a proportional relationship between the mRNA and protein levels does not exist and what functional significance the altered mRNA or protein levels may have in lung tumorigenesis. Certain MAP3K8 binding partners can prevent proteolytic degradation of the protein, but the binding also inactivates the protein. Release to an unbound state activates the protein and its downstream pathways, but unbound MAP3K8 is also quickly degraded (Belich, 1999; Waterfield, 2003). Due to this relationship, it is possible that the MAP3K8-highly transcribing cell lines, such as A549, may be constantly translating and turning over unbound, active MAP3K8. However, since the protein is rapidly degraded, it never reaches a detectable level identified through standard Western blot techniques. It is tempting to speculate that such a continuous pool of transcribed, translated, and activated MAP3K8 may be an oncogenic alteration significant to a subset of lung cancers. However, until further research is performed it remains unclear why such discrepancies exist between the observable MAP3K8 mRNA and protein levels in lung cancer cell lines.
It has been demonstrated that MAP3K8 expression contributes to tumorigenesis.
Transfection and over-expression of the gene into SHOK and NIH3T3 cells induces cellular transformation, and transfection of the gene into T cell lymphocytes induces cell cycle progression and proliferation (Miyoshi et al., 1991; Aoki et al., 1993; Chan et al.,
1993; Velasco-Sampayo et al., 2001). To examine the functional activity in lung cells, we transfected the 9HTE cell line with a wildtype and an oncogenic mutant form of 101
MAP3K8. Unexpectedly, transfection of both the wildtype and mutant MAP3K8 resulted in slowed proliferation of the cells. The 9HTE-MUT but not the 9HTE-WT cells appeared to slow proliferation through G0/G1 arrest (Figure 8). Similar proliferation inhibition was reported after transfection of MAP3K8 into mouse embryonic fibroblast
(MEF) cells (Lund et al., 2002). ). However, following knockout of the Cdkn2a gene, the gene that encodes the p16 tumor suppressor, the MEF cell growth inhibition was released and the cells increased proliferation. Similarly, transfection of MAP3K8 into the tumorigenic CTLL-2 cells also induced proliferation and cell cycle progression (Valesco-
Sampayo et al. 2001). Since the 9HTE-WT and 9HTE-MUT cells mimicked the growth of the Cdnk2a-positive MEF cells, loss of the p16 tumor suppression pathway or other tumor suppressor pathways may be prerequisite steps for MAP3K8 contribution to lung tumorigenesis. 102
Figure 10. 9HTE growth curve analysis 2. A second growth curve was generated with the 9THE transfectants. However, these curves were generated using 250,000 cells plated onto 10cm diameter plates. All the samples were done in triplicate for each timepoint.
In order to test the possibility that tissue culture factors outside of proliferation were influencing the growth curve, a second curve was generated over twelve days after plating 250,000 cells per 10 cm plate. This differed from the 1000 cells per 3 cm plate in the first curve. Interestingly, the second curve did not show an increased number of cells in the 9HTE-CMV cells, but instead showed similar growth rates for all three cell lines. It is unclear why this difference occurred between the two growth curves. It is possible that that the plating efficiency of the cells may have influenced the growth curve of the transfected cell lines. Likewise, since the 3 cm plates reach confluency by day 8 and the
10 cm plates did not reach confluency until day 12, the growth inhibition in the first curve may have been due to a combination of MAP3K8 activity and other factors associated with cellular contact. However, no experiments have been done to test either of these possibilities.
MAP3K8 also activates apoptosis in non-transformed HEK293 and REF52 fibroblast cells. We investigated if this pathway was utilized in the 9HTE-WT and 9HTE-
MUT cells to account for the different cell cycle profiles but similar growth rates. Neither the TUNEL assay nor a caspase activity assay demonstrated differences in apoptotic levels among the transfected lung cell lines. However, the 9HTE cells were stably transfected with an expression vector with a strong promoter. The report identifying
MAP3K8-induced apoptosis used an inducible expression vector (Patriotis et al., 2001). ).
It is possible that the transient induction of MAP3K8 in those cells activated the apoptotic machinery as a non-specific result of over-expression. Alternatively, the Caspase-9 103 activation by MAP3K8 may instead be an intrinsic function of the protein that requires specific cellular environments; environments that exist in HEK293 and REF52 cells but not in 9HTE cells. In either case it does not appear that the decreased proliferation resulting from transfection of wildtype MAP3K8 into 9HTE cells was the result of apoptotic induction.
The data presented here support a role for MAP3K8 in lung cancer tumorigenesis through altered transcriptional and translational regulation. Since cancer is a multistep process, MAP3K8 activation is not sufficient to induce cellular transformation alone.
Given the apparent relationships with Cdkn2a/p16 and MAP3K8-induced growth suppression in non-transformed cells, increased expression of the gene may be a contributing factor to tumorigenic progression after other alterations occur in the cell.
Upon loss of growth arresting and tumor suppressive genes, increased expression and increased activation of MAP3K8-induced pathways may facilitate cellular growth and transformation. Since breast cancers also demonstrated an increased expression of
MAP3K8 when compared to the surrounding normal breast tissue, it is possible that the increased transcription of the gene is a characteristic alteration of multiple cancer types.
However, since the protein and mRNA levels do not correlate in lung cancer cell lines, other factors such as protein stabilization and inactivation need to be examined to better understand the role of the MAP3K8 protooncogene in lung tumorigenesis. 104
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Chapter 6
MAP3K8 Expression in 9HTE Cells and the Effect on
Downstream Pathways 108
Introduction
The MAP3K8 protooncogene was not found to be frequently mutated in lung cancers, but did display alterations at both the mRNA and protein levels within the different tumor cell lines. The alterations varied from nearly absent mRNA expression with high protein expression to greater than 50-fold normal lung cell mRNA expression without detectable protein expression. The production of the two different isoforms also varied among the cell lines, although the SCLC cell lines tended to more frequently express both isoforms if the protein was at sufficient levels high enough for detection.
While the significance of the expression levels is not understood, it was demonstrated that increased expression of the wildtype and mutant forms of the gene had a phenotypic effect on the 9HTE cells without exposure to extracellular stimuli, suggesting that MAP3K8 expression is sufficient to induce different cellular activities. However, only the mutated form was able to induce changes in the cell cycle profile exhibited in the FACS analysis.
This suggested that mutated and wildtype MAP3K8 may have different activities, due to the change in amino acid sequence, activating different pathways but inducing similar responses in the growth curve.
Signals conveyed through the MAP kinase pathway and other signal transduction pathways ultimately induce differential activation of the nuclear transcription factors regulating mRNA expression. MAP3K8 influences activation of a variety of transcription factors, including NFAT, AP-1, CREB, and NF-kB. Through activation of these factors, multiple genes are transcribed that are influential in cellular growth and survival such as
IL-2 and COX-2 (Ballester et al., 1997; Tsatsanis et al., 1998; de Gregorio et al., 2001; 109
Eliopoulos et al., 2002). Both wildtype and mutant MAP3K8 were able to induce activation of these pathways under unstimulated conditions, but activation by extracellular stimuli such as LPS, induced MAP3K8 activation of downstream transcription factors and promoted increased expression of these genes to a much greater extent (Tsatsanis et al.,
1998; de Gregorio et al., 2001; Eliopoulos et al., 2002). These results suggest that
MAP3K8 serves as a regulator in response to extracellular stimuli, but also has intrinsic activities that may not require activation from upstream stimuli.
Similarly, MAP3K8 phosphorylates many downstream targets to activate these different pathways and induce alterations in transcription of the genes. However, there are multiple phosphorylation sites on proteins with different regulatory roles and, each of these can influence different protein and cellular functions. For example, the signal transduction kinase PKCa, is regulated by diacylglycerol (DAG), and is activated by phosphorylation on the activation loop at threonine 497 (T497). Phosphorylation at this site allows catalytic activation by DAG. In addition, a threonine at amino acid 638 (T638) complements the T497 phosphorylation, but has no functionality in the catalytic activity of the protein. Instead, phosphorylation at T638 prevents dephosphorylation and inactivation of the enzyme, stabilizing the active conformation (Bornancin and Parker,
1996). Many proteins are regulated by phosphorylation and differential phosphorylation of the protein can ultimately affect the downstream pathways and regulation of transcription factors.
Since the transfection of the wildtype and mutant MAP3K8 influence cellular growth characteristics, we examined what factors participated in these characteristics through transcription factor array analysis and site specific phosphorylation array analysis 110 to characterize any alterations. Here we show that both wildtype and mutant MAP3K8 activate multiple downstream transcription factors in non-stimulated 9HTE cells including known downstream transcription factors such as NFAT. The array also identifies previously unknown targets of MAP3K8 such as the Brn-3 and MRE transcription factors.
Site-specific phosphorylation analysis of the 9HTE transfected cell lines did not demonstrate increased phosphorylation of known substrates such as MEK-1 or MKK3 at the sites analyzed, but did identify increased phosphorylation of AMP-activated protein kinase a (AMPKa) and PKCa/b. While activation of these different pathways does not fully explain the cell cycle alterations demonstrated in the delayed proliferation and
G0/G1 arrest, they do demonstrate that overexpression MAP3K8 influences regulation of cellular proteins and pathways that play roles in growth and tumorigenesis.
Materials and Methods
Nuclear Lysate Extraction: 9HTE transfected cells (9HTE-CMV, 9HTE-WT, and 9HTE-
MUT as previously described) were grown in DMEM media supplemented with 10%
Fetal Bovine Serum and 400 ug/mL G418 antibiotic. The nuclear lysate was extracted using the Nuclear Extraction Kit (Panomics, Redwood City, California) according to the manufacturer’s protocol. The protein concentration was quantified using the BCA Protein
Assay Kit (Pierce, Rockford, IL) in triplicate samples according to the manufacturer’s protocol. The samples were used immediately after extraction.
Transcription Factor Array: The TranSignal Protein/DNA Array I (Panomics, Redwood
City, CA) was used for the transcription factor array analysis in duplicate analysis for each 111 cell line. The array measures the activity of 54 different known transcription factors. The array only measures active nuclear proteins that are able to bind DNA and is, therefore, not a measure of protein quantity, but a qualitative measure of the relative activity of the transcription factors. The array was developed according to the manufacturer’s protocol.
In brief, 20 ug of nuclear extract from each sample was incubated with and bound to the
TransSignal Probe Mix. The samples were run on a 2% agarose gel in 0.5X TBE Buffer at 4 degrees C to separate the bound probe from the unbound probe. The gel containing the bound probe was excised and the DNA/protein complexes were isolated from the gel.
The probed DNA was separated from the protein and hybridized to the transcription factor array membrane (Figure 1). The membrane was washed, blocked, and incubated with a streptavidin-HRP conjugate. The membrane was washed again and incubated with the detection buffer and working substrate solution provided with the kit. The array was exposed to film at varying time points from 5 second to 10 minutes. Array analysis was performed by comparing exposed film that displayed matching intensities of the control signals on the edges of the membrane. The control signals, as well as the transcription factor signals, were measured by densitometry using the ImageJ densitometry program available at the National Cancer Institute website (http://rsb.info.nih.gov/ij/). The total control signal intensity for each array was measured and a ratio was calculated among the three arrays to normalize the intensities. Each transcription factor has four spots for each signal. Two spots at a 1.0X concentration and two spots at a 0.1X concentration. Only the 1.0X concentration was used for analysis because the relative intensities of the 1.0X and 0.1X did not display a 10:1 ratio. The densitometry average of the two signals for 112 each array was calculated as Relative Densitometry Units. The Mean +/- Standard Error of the Mean (SEM) was calculated for the two arrays and graphed.
PCR Amplification: Products were amplified from cDNA reverse transcribed from the transfected cell lines 9HTE-CMV, 9HTE-WT, and 9HTE-MUT using the Biolase
Polymerase (Bioline USA Inc., Randolph, MA) according to manufacturer’s instructions.
The following primer pairs were used for each gene _-Internexin (5’-
GAGGAACACCAAGAGTGAGATGGCAC-3’/ 5’-
GGCAGGTGTTATAGTTCAGGAATAGGC-3’), Bcl-2 (5’-
CGACAACCGGGAGATAGTGATGAAG-3’/ 5’-
GCTGGCTGGACATCTCGGCGAAGT-3’), IP3RI (5’-
CAGCGTGGTCATAGGTGACAAGGTG-3’/ 5’-
CAACCTACTTCGAGAGGCGTCCTGA-3’), MTI (5’-
ATGGACCCCAACTGCTCCTGCG-3’/ 5’-GCACTTCTCTGATGCCCCTTTGCA-3’),
MTII (5’-GATCCCAACTGCTCCTGCGCCGC-3’/ 5’-
CTGCACTTGTCCGACGCCCCTTTG-3’), and GAPDH (5’-
GAAGGTGAAGGTCGGAGTCAACGG-3’/ 5’-
CACTTGATTTTGGAGGGATCTCGCTC-3’). The products were run in a 2% NuSieve
Gel (BioWhittaker Molecular Applications, Rockland, ME) and visualized under UV light.
Phosphorylated Protein Array: The phosphoamino acid analysis of various proteins was performed with the Kinetworks Phospho-Site Screen KPSS-4.0 (Kinexus Bioinformatics 113
Corporation, Vancouver, British Columbia, Canada). Total cellular lysate containing 500 ug of protein (1 mg/mL) was isolated from the transfected cell lines 9HTE-CMV, 9HTE-
WT, and 9HTE-MUT and quantified by triplicate analysis of through the BCA Protein
Assay Kit (Pierce, Rockford, IL). The samples were prepared according to the manufacturer’s suggestions and analyzed at the Kinexus Bioinformatics Corporation. The array analyzed proteins with 39 phosphorylation site specific antibodies. The bands were visualized and the area under the band’s intensity profile curve was reflective of the trace quantity of phosphorylated protein present. The relative intensities were measured as counts per minute (CPM) and the CPM for each band was normalized based on a coefficient value established between the different samples. The 9HTE-WT and 9HTE-
MUT results were graphed as a percentage increase or decrease of the relative 9HTE-
CMV CPM values. Percent differences greater than 20%, either increasing or decreasing, are considered by Kinexus Bioinformatics Corporation to be significantly different. 114
Figure 1. Illustration of the TranSignal Protein/DNA procedure from Panomics Protocol. Active transcription factors bind the labeled probe DNA. After a series of separation steps this DNA is hybridized to the array membrane, incubated with a streptavidin-HRP conjugate, treated with a chemiluminescent substrate, and measured for signal intensity. 115
Results:
Nuclear lysate from each of the transfected cell lines, 9HTE-CMV, 9HTE-WT, and 9HTE-MUT was isolated for analysis of activated transcription factors. The
TranSignal Protein/DNA array I measures only active transcription factors and is used for a qualitative analysis of this activity. We performed the analysis on two different arrays for each transfected cell line using fresh nuclear lysate each time. A comparison of one set of arrays is illustrated in Figure 2. The signals along the bottom and right sides of the arrays are control spots used to normalize intensities and exposure times. The average densitometries of the spots were calculated and nine transcription factors were differentially activated among the different cell lines (Figure 3). These transcription factors were: Brn-3, CBF (CAAT Binding Factor), c-Myb, ERE (Estrogen Receptor),
ETS1/PEA3, GATA, NFATc, NF-E2, and MRE (Metal Response Factor). When compared to 9HTE-CMV cells, Brn-3 was greatly activated in both the 9HTE-WT and
9HTE-MUT cell lines. CBF was only activated in the 9HTE-WT cells, and NFAT appear to be activated only in the 9HTE-MUT cells. For c-Myb, neither the 9HTE-WT nor
9HTE-MUT were different from the 9HTE-CMV cells, but they were different from each other. The 9HTE-WT cells induced activation of the c-Myb transcription factor and the
9HTE-MUT cells decreased activation of c-Myb. Both the ERE and ETS1/PEA3 transcription factors did not show any activity in the 9HTE-CMV cells, but displayed induced activation in both the 9HTE-WT and 9HTE-MUT cells. Finally, GATA, NF-E2, and MRE displayed decreased activation in the 9HTE-WT and 9HTE-MUT cells. While the decrease in NF-E2 was marginal, there was a large decrease in activation of GATA and MRE. Transfection of the MAP3K8 gene has been associated with activation of the 116 transcription factors AP-1, CREB, E2F, and NF-kB contained on the array. Calculation of the Mean +/- SEM found no difference between activation of these transcription factors and transfection of the wildtype or mutant MAP3K8, although the AP-1 transcription factor did appear to demonstrate a trend towards increased activity from wildtype and mutant MAP3K8.
9HTE-CMV
9HTE-WT 117
9HTE-MUT
Figure 2. TranSignal Protein/DNA array. The arrays aligned from top to bottom are 9HTE-CMV, 9HTE-WT, and 9HTE-MUT. The signals at the bottom and right hand sides of each array are control signals and do not correspond to any transcription factor. The nine boxes outlined in green are the transcription factors that display differential activation among the cell lines. Listed from Top to Bottom and Left to Right are: Brn-3, CBF, c-Myb, ERE, ETS1/PEA3, GATA, NFATc, NF-E2, and MRE. 118
Figure 3. MAP3K8 TranSignal Array Analysis. The chart displays the mean relative densitometry units (+/- SEM) between the two arrays analyzed for each cell line. Below the chart are arranged individual signal spots from the arrays that correspond to the activated transcription factors. Transcription factors marked with an * indicate that there was no difference between the activity of the transcription factors among the three cell lines.
In order to demonstrate a proof of principle for the transcription factor array, several genes were chosen for RT-PCR amplification. These genes contain response elements in their promoters corresponding to several of the transcription factors.
Activation of the transcription factors could be reflected in downstream transcription of these genes as demonstrated by RT-PCR (Figure 4). Both _-Internexin and Bcl-2 promoters contain Brn-3 binding motifs and were transcriptionally activated by Brn-3
(Budhram-Mahadeo et al., 1995, Ensor et al., 2001). Amplification of the _-Internexin gene clearly shows an increased level in the 9HTE-WT and 9HTE-MUT cells, the cells that display activated Brn-3, compared to the 9HTE-CMV cells. However, only the
9HTE-MUT cells appear to increase transcription of the Bcl-2 gene. Activation of NFAT by MAP3K8 has been demonstrated by different groups and has been supported by the transcription factor array (Tsatsanis et al., 1998, de Gregorio et al., 2001). The inositol
1,4,5-trisphosphate receptor type 1 (IP3RI) was transcriptionally activated by NFATc
(Graef et al., 1999). The activation of NFATc in the 9HTE-MUT cells was clearly distinguishable from the 9HTE-CMV cells, whereas the 9HTE-WT cells appeared to activate NFATc higher than 9HTE-CMV but were within the range of the standard error.
However, both the 9HTE-WT and the 9HTE-MUT demonstrated a clear increased expression of IP3RI mRNA relative to 9HTE-CMV and the Metal Response Ewlement
Transcription Factor (MRE) was inactivated in both the 9HTE-WT and 9HTE-MUT cells 119 compared to the 9HTE-CMV. MRE transcriptionally activated the metallothionein genes
MTI and MTII (Foster and Gedamu, 1991; Koizumi et al., 1999). RT-PCR analysis showed that only the MTII mRNA was decreased in these cells lines. The MTI transcriptional regulation appeared to be unaffected by MAP3K8.
Figure 4. RT-PCR amplification of the transcription factor regulated genes. The genes (listed on the left) were amplified from each of the cell lines. The corresponding transcription factor that transcriptionally regulates the gene is listed on the right.
A phospho-site specific protein array was also analyzed for the different transfected cell lines to measure the relative site-specific phosphorylation of a variety of 120 different proteins. The analysis was performed by the Kinexus Bioinformatics
Corporation. We isolated the total cellular fraction of each of the cell lines and analyzed the differential phosphorylation of the proteins within the fraction. The intensity of the band was measured as counts per minute (CPM) and each array was normalized by
Kinexus Bioinformatics Corporation so that the Normalized CPM for the 9HTE-WT and
9HTE-MUT was expressed as a percent difference of the 9HTE-CMV cells. A 20% difference in phosphorylation was accepted as significantly different by Kinexus
Bioinformatics Corporation. Figure 5 displays the results of the visible results of the arrays. 121
Figure 5. Kinexus Phospho-Site Screen (KPSS-4.0) Analysis. The arrays aligned from top to bottom are protein lysates from cell lines 9HTE-CMV, 9HTE-WT, and 9HTE- MUT. The bands correspond to a site-specific phosphorylated protein. The numbers in red to the left of the bands indicate which protein the band corresponds to as listed in Table 1. 122
Table 1. Site-specific phosphorylation data. The data presented here lists the numeric results from the Kinexus Array (4.0). The columns list from left to right the name of the phosphorylated protein; a commonly used abbreviation for the protein; the site-specific amino acid that was phosphoylated and recognized in the array; the number (in red) that corresponds to the band in Figure 4; and the normalized counts per minute of intensity read for each band that corresponds the relative amount of phosphorylated protein. ND= not detectable. 123
Figure 6. Relative MAP3K8-induced Site-specific Phosphorylation. The graph illustrates the relative % difference in counts per minute (CPM) the 9HTE-WT and 9HTE-MUT displayed in the phosphorylated proteins compared to the 9HTE-CMV cells. The 9HTE- CMV readings are established as the base line and are given a value of zero. A 100% difference in either direction is equivalent to a 2-fold difference in CPM. Kinexus Bioinformatics Inc. considers a 20% difference to be significant.
Of the proteins analyzed, 19 were measurable on the arrays (Figure 5, Figure 6,
Table 1). Several of the proteins, 4E-BP1, Lyn, and Raf had different isoforms recognized by the antibody and were labeled by their molecular weight in parentheses such as 4E-BP1(16) and 4E-BP1(17). In other cases, two antibodies recognized the same protein, but different phosphorylation sites. The proteins that demonstrated a significant 124 difference in phosphorylation (+/-20% of the 9HTE-CMV levels) were 4E-BP1, AMPK·,
Cdk1 Y15 (only significantly different in the 9HTE-WT, Lyn Y507 (44), MKK6 (only significantly different in 9HTE-WT; the 9HTE-MUT levels were not detectable), mTOR, p70S6K (both the T389 and the T421/S424 phosphorylation site), PDK1 (only significantly different in the 9HTE-WT), PKC_ /b, PKC z/l, PRK2 (only significant different in the 9HTE-MUT), and Rb. The arrays also displayed a number of unlabeled proteins recognized by the different antibodies but classified as “unknown proteins” by the Kinexus Bioinformatics Corporation (data not shown). It is unclear if these protein were a variant form of one of the known proteins that was not recognized by the analysis or if the band corresponded to non-specific binding of one of the antibodies and existed as an experimental artifact. The KPSS-4.0 array was designed to recognize a number of additional proteins that are phosphorylated by the upstream activation of MAP3K8 but did not show detectable levels recognized in any of these arrays. These proteins were: Erk-1/2
T202/Y204, Erk-1/2 S217/S221, IkB-_ S181, IkB-_ S180, MEK-1/2 S217/S221, and p38
T180/Y182.
Discussion
The MAP3K8 protooncogene conveys oncogenic properties by inducing cellular transformation and growth proliferation in certain cells through activation of cellular pathways, such as the MAP Kinase signal transduction cascade, or activation of the anti- apoptotic and cellular survival pathways, such as the NF-kB signalsome. In contrast, activation of MAP3K8 also regulates tumor-suppressive activities, such as growth arrest and Caspase-9 induced apoptosis. Activation of these opposing pathways suggests that 125 specific cellular contexts determine the particular activity MAP3K8 induces. A variety of different scenarios may be associated with the selection or preference of one pathway over another including cell type, loss or gain of function in other proteins, variable binding partners, and different extracellular stimuli. We recently discovered that in unstimulated
9HTE cells, transfection of both the wildtype and oncogenic mutant forms of MAP3K8 slowed proliferation of the cells in a non-apoptotic manner. The mutant gene arrested the cells in the G0/G1 phase of the cell cycle whereas the wildtype did not. These results suggested that the wildtype and mutant genes had overlapping activities, but may vary in the different pathways in which they participate to elicit these activities.
Since MAP3K8 has been notably associated with many transcription factors such as AP-1, CREB, E2F, NFAT, and NF-kB, we decided to examine activation of these transcription factors and others through analysis of a signal transduction factor array
(Tsatsanis et al., 1998(1); Tsatsanis et al., 1998 (2); de Gregorio et al., 2001; Eliopoulos et al., 2002; Lin et al., 1999; Valesco-Sampayo et al., 2001; Kane et al., 2002). Nuclear lysate from each of the transfected cell lines was isolated and immediately incubated with the labeled probe mix containing various DNA binding domain motifs for the different transcription factors. After separation from the activated nuclear proteins the probe was hybridized to the transcription factor membrane and developed. The procedure was performed in duplicate, each with fresh nuclear lysate, to analyze for differences in transcription factor activation.
A variety of transcription factors were activated in the cells, but 9 were differentially activated among the transfected cell lines. Only one transcription factor activated in the array, NFATc, had been previously shown to be activated downstream of 126
MAP3K8 (Tsatsanis et al. 1998). This data supported the previous findings implicating
NFAT activation in the MAP3K8 pathways and indicated that NFATc was activated by
MAP3K8 in lung cells. In order to demonstrate a proof of principle for the array, the
IP3RI gene, a gene that is transcriptionally activated by NFAT, was chosen as a subject gene for analysis. PCR amplification indicated that the IP3RI gene was expressed at higher levels in both the 9HTE-WT and 9HTE-MUT cells even though the 9HTE-MUT cells were the only cells displaying a difference from the 9HTE-CMV cells in NFAT activity in the array. However, multiple transcription factors exist to regulate genes.
Therefore, these results were not conclusive that NFATc activation caused increased expression of the IP3RI gene, but they do support that conclusion.
Two transcription factors ERE and ETS1/PEA3 were induced by the 9HTE-WT and 9HTE-MUT cells, and two proteins, the zinc-finger transcription factor GATA and the stress-associated transcription factor MRE, were both decreased in activity in the
9HTE-WT and 9HTE-MUT cells compared to the 9HTE-CMV cells in the transcription factor array analysis. PCR amplification of MRE transcriptionally activated genes, MTI and MTII, demonstrated activation of the MRE transcription factor in the cell lines.
Interestingly, the decreased activity of MRE appeared to have no effect on the transcription of MTI. But MTII transcription was severely attenuated in these cells, supporting MAP3K8-induced inactivation of MRE in lung cells. In addition, the NF-E2 transcription factor also decreased slightly, but significantly, in activity in the 9HTE-WT and 9HTE-MUT cell lines.
One transcription factor, c-Myb, the cellular homologue to the viral myeloblastoma oncogene (v-Myb) did not display differences between either the 9HTE- 127
WT or 9HTE-MUT cells from the 9HTE-CMV cells, but rater, displayed differences between the 9HTE-WT and 9HTE-MUT. It appeared that the 9HTE-WT increased activity of c-Myb but 9HTE-MUT decreased activity of c-Myb. To date no relationship between MAP3K8 and c-Myb has been described, but the differential activation of the protein between the wildtype MAP3K8 and the mutant MAP3K8 suggests that carboxy- terminal mutations, which were believed to activate the MAP3K8 protein, may also induce different functions on the mutant protein compared to the wildtype. This is further supported by the activation of the general transcription factor CBF in the 9HTE-WT but not 9HTE-MUT cells.
Finally, the Brn-3 transcription factor was greatly activated in both the 9HTE-WT and 9HTE-MUT cells. Brn-3 is a family of transcription factors belonging to the POU gene family (named for similarity to the Pit, Oct, Unc transcription factors). Among other cell types, the Brn-3 transcription factors are expressed during nerve and neuroendocrine cell development. They are able to transform rat embryonic fibroblast cells (REF) when co-expressed with Ha-Ras (Theil et al., 1993). Interestingly, Brn-3 interacts with the estrogen receptor transcription factor increasing transcriptional activity of the ERE upon interaction (Budhram-Mahadeo et al., 1998). The relationship may explain the increased activation of ERE observed in only the 9HTE-WT and 9HTE-MUT cells. Increased activation of Brn-3 is mechanistically linked to tumorigenesis through increased expression and translation of the anti-apoptotic Bcl-2 protein (Chiarugi et al., 2002).
However, wildtype p53 interacts directly with Brn-3 and antagonizes its ability to transactivate Bcl-2, a relationship that is lost in p53 mutants (Budhram-Mahadeo et al.,
1999). Likewise, Brn-3 abolishes p53 transcriptional activation and protein expression of 128 the pro-apoptotic protein Bax (Budhram-Mahadeo et al., 2002). Brn-3 and p53 cooperate to induce transcriptional activation of the cyclin kinase inhibitor p21CIP1/Waf1 and induce cell cycle arrest (Budhram-Mahadeo et al., 2002). This relationship may explain the
MAP3K8-induced growth suppression in 9HTE cells. 9HTE cells are immortalized, but non-transformed bronchial epithelial cells. The p53 genotype of 9HTE cells is unknown.
But if the gene is not mutated, activation of Brn-3 by MAP3K8 may result in functional p53 protein cooperation with Brn-3 to induce upregulation of p21CIP1/Waf1 and arrest the cells. We amplified two Brn-3 regulated genes a-Internexin and Bcl-2 to determine if their expression was increased to coincide with increased Brn-3 activity in the cells. a-
Internexin demonstrated high levels of transcriptional expression in both the 9HTE-WT and 9HTE-MUT cells compared to the 9HTE-CMV cells, supporting activation of Brn-3 in the regulation of the gene. The Bcl-2 gene did not display increased transcriptional activation in the cell lines. This fits within the paradigm of non-mutated p53 in the cells.
Wildtype p53 has never been shown to affect a-Internexin expression, but will bind with
Brn-3 to inhibit increased Bcl-2 expression. Since p53 is often mutated in lung cancers, as well as many other cancers, MAP3K8-induced activation of Brn-3 may have a pro- oncogenic effect in these cells, through Bcl-2 upregulation, and contribute to the tumorigenic initiation and progression after mutation of p53 occurs.
In order to complement the transcription factor array, we analyzed the site- specific phosphorylation of a number of proteins using the Kinetworks Phospho-Site
Specific Protein Array (KPSS-4.0). The array measures the amount of phosphorylation of a given protein at a specific site using antibodies specific to the phosphorylated protein.
Total cellular lysate is run through a gel in 18 different lanes and probed with labeled 129 antibodies. The signals received from the bound antibodies are measured as counts per minute (CPM) and these measurements are normalized among the arrays to provide a means to measure the relative amount of phosphorylated protein. Kinexus Bioinformatics
Corporation performed the analysis using a variety of different antibodies. Figure 5 illustrates the array data and Figure 6 and Table 1 compile the data with the normalized
CPM. The 9HTE-CMV were used as the control and the values collected from these cells were established as the base line values.
Of the proteins examined, 19 were expressed at detectable levels. These were graphed as % Difference From 9HTE-CMV CPM (Figure 6). Interestingly, only one known downstream protein of MAP3K8, MKK6, was shown to be phosphorylated, but only in the 9HTE-WT cells. MKK6 phosphorylation was not detectable in the 9HTE-
MUT cells. MKK6 phosphorylation at S207 was increased 83% over the 9HTE-CMV levels, but this phosphorylation was not reflected in p38 phosphorylation on T180/Y182, the protein that lies downstream of MKK6. Even though the relative amount of MKK6 phosphorylation was increased in the 9HTE-WT cells, both cell lines showed low CPM in the array.
None of the other known downstream proteins of MAP3K8 (Erk-1/2, IkB-a, IkB- b, or MEK-1/2) displayed measurable site-specific phosphorylation on the array. The previous studies that demonstrated that MAP3K8 activated these proteins did not measure site-specific phosphorylation. It is possible that other sites on these proteins were phosphorylated but were not measured in this array. Another possibility is that either the lung cell environment or the lack of an activating stimulus was not conducive to MAP3K8 activation of these proteins on the sites measured. In either case, this data is relevant and 130 important because it helps to delineate the downstream site-specific proteins that
MAP3K8 affects.
The array did identify several novel pathways that have never been associated with
MAP3K8. Wildtype and mutated MAP3K8 appeared to inhibit phosphorylation of one set of proteins involved in the same pathway. 4E-BP1, p70S6K, and mTOR all displayed decreased site-specific phosphorylation in the 9HTE-WT and 9HTE-MUT cells (one exception was that mTOR was decreased 42% in 9HTE-WT cells, but increased 69% in
9HTE-MUT cells). These proteins signal in a pathway that regulates mRNA translation, protein synthesis, cellular growth and development. Activated mTOR phosphorylates 4E-
BP1 at S65, releasing the elongation factor elF4E and allowing mRNA translation; and hyperphosphorylates p70S6k at multiple sites, allowing the protein to activate the 40S ribosomal protein, resulting in increased protein synthesis, cellular growth and differentiation (Dennis et al., 1996; Nave et al., 1999; Gingras et al., 2001; Huang et al.,
2002). Both these pathways are important in cell growth and tumorigenesis and inhibition of these pathways slows progress through the cell cycle. However, it is not yet known if
MAP3K8-induced inhibition of these pathways influenced 9HTE cell growth.
Since MAP3K8 is a kinase, it is interesting that transfection of the gene resulted in a decrease in the phosphorylation of these proteins. One possible explanation for this is that high levels of MAP3K8 may be interfering with an activator of mTOR. One known binding partner for MAP3K8 is AKT, a protein that lies upstream of mTOR S2448 phosphorylation (Kane et al., 2002; Nave et al., 1999). While purely speculative, it is possible that the high level of MAP3K8 in the 9HTE cells sequestered the AKT protein and inhibited downstream activation of mTOR, 4E-BP1, and p70S6K (Figure 7). 131
Regardless of the means, the decreased phosphorylation of all three proteins in this pathway indicates that MAP3K8 influences the phosphorylation status of the proteins in the mTOR pathway.
Figure 7 (above). Illustration of the proposed MAP3K8-induced inhibition of the mTOR signaling pathway. MAP3K8 transfected cells displayed reduced site-specific phosphorylation of the mTOR, 4E-BP1, and p70S6K proteins, all of which are in the same pathway. It is unknown how MAP3K8 altered these proteins, but it is known that MAP3K8 participates in certain pathways with Akt, a known activator pf mTOR and it is possible this pathway was influenced, a;though no data exists to ascertain this relationship.
Other proteins such as AMPKa, a protein regulated by cellular ATP levels and glucose uptake, and PKCa ,a protein activated by the second messenger diacylglycerol, 132 were greatly phosphorylated in both the 9HTE-WT and 9HTE-MUT cells. They displayed both high CPM and a high percentage difference from the 9HTE-CMV cells.
These proteins are important in many cellular signaling pathways. If MAP3K8 is an activator of these pathways, its relevance is more important in cellular function and activity than previously suspected. The precise role it plays in the phosphorylation of these proteins and their downstream pathways needs to be further investigated.
There are several other proteins that displayed marginal changes in phosphorylation in the array and may or may not be significantly associated with
MAP3K8 activity and cell growth. It is interesting to note that the PKC related kinase 2,
PRK2, appeared to have high levels of phosphorylation only in 9HTE-MUT cells. Also, the Rb protein appeared to be phosphorylated in 9HTE-MUT cells but dephosphorylated in 9HTE-WT cells. These data provide additional support for the possibility that the mutated MAP3K8 gene may have intrinsic activities that contribute to tumorigenesis that the wildtype gene does not possess. Regardless, both the wildtype and mutant genes appear to play significant roles in activation of a larger battery of proteins than previously thought.
Taken together, these data demonstrate that non-stimulated MAP3K8 is active within the 9HTE lung cells. We were not able to show increased phosphorylation in the
MAP kinase pathways. This may be due to other site-specific phosphorylated amino acids on the protein which were not analyzed in the array, or it may indicate that MAP3K8 requires a stimulus or release from an inhibitor to phosphorylate and activate these proteins. We do show that MAP3K8 participates with other proteins such as Brn-3, mTOR, and PKCa/b; all of which have important roles in tumorigenic development. 133
Altered MAP3K8 regulation of these pathways, along with other tumorigenic alterations such as p53 mutation or loss of p16, may influence regulation of cellular growth and help progress the cell towards transformation and tumor development. 134
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Chapter 7
Conclusions 138
As stated in the beginning of this report, cancer is a complex disease that develops as a multi-step process. It has both environmental and genetic components contributing to its development. Lung cancer, in particular, is primarily an environmentally induced disease, making the research of lung cancer an appropriate field of study for environmental health. A number of compounds are linked to lung cancer development, but cigarette smoke is by far the most potent causal agent for this disease, followed by asbestos, radon, and air pollution. All have a contributing role in lung tumorigenesis and influence the cellular activities to eventually lead to lung cancer. This dissertation sought to analyze the role of MAP3K8 in lung tumorigenesis in order to gain a better understanding of the biology of MAP3K8 signal transduction pathways in lung cancer and to improve on the basic understanding of tumorigenic pathways.
Many alterations occur during tumorigenic development that complement increased growth, proliferation, survival, and differentiation allowing a normal cell to develop into a cancer cell. These alterations occur through various mechanisms such as genetic mutations which alter the protein function, epigenetic alterations which change the transcriptional regulation of a gene, loss of a gene, amplification of a gene, and post- translational modifications of the protein. Regardless of the means by which the alterations occur, it is ultimately the outcome of such alterations that affects cellular activities.
The MAP3K8 protooncogene was introduced in this dissertation as a gene that produced a protein having roles in pathways that regulated cellular growth, proliferation, and death. Originally identified as a mutant gene able to transform SHOK cells, the 139
MAP3K8 gene was later characterized as one of the Mitogen Activated Protein Kinase
Kinase Kinase (MAP3K) signal transduction proteins, which regulate the activity of many signaling pathways. Prior to the reports presented here, no mutation of human MAP3K8 had been found to occur in a primary human tumor. However, the isolation of a mutated
MAP3K8 from a human lung adenocarcinoma supported the hypothesis that MAP3K8 might be targeted for mutation in lung tumorigenesis. Since the functional activity of a protein can be influenced in many ways outside of mutation, a broader hypothesis was developed to examine the role of MAP3K8 in lung tumorigenesis. This hypothesis included experimental study of three primary areas of MAP3K8 biology: first, a mutational analysis of MAP3K8 in lung cancer; second, an analysis of MAP3K8 transcriptional and translational expression in lung cancer cell lines; and third, an analysis of the functional effects of transfection of wildtype and mutant MAP3K8 into non- transformed lung cells.
Mutational analysis of the gene began with DNA isolated from a primary lung adenocarcinoma named L-41. The DNA isolated from this tumor transformed NIH3T3 cells. Series of additional experiments ultimately identified the MAP3K8 gene as the transforming gene from the DNA. Sequence analysis of cDNA libraries generated from the NIH3T3 transfected cells demonstrated that the MAP3K8 gene was mutated at the
3’end of the open reading frame resulting in a carboxy-terminal altered protein.
Amplification of the DNA from the primary tumor confirmed this mutation existed in the primary lung tumor DNA, thus identifying the first MAP3K8 mutation existing in a primary human tumor. The transformation studies which followed confirmed that even though the mutation was different from the original thyroid tumor mutant and the 140 mutations identified in the MAP3K8 rat homologue, Tpl-2, this lung tumor mutant was able to induce tumors in nude mice at a high frequency. This supported data indicating that alterations at the carboxy-terminus confer oncogenic activities to the gene.
A search for additional mutations of the gene in lung cancer cell lines through
3’RACE analysis did not identify additional mutations occurring in the 3’ end of the gene.
This was complemented by PCR-SSCP screening of the open reading frame. PCR-SSCP is an excellent method to search for point mutations and small mutations in the DNA because the migration of the amplified product in the gel is highly sensitive to these changes. However, the procedure does not identify every mutation and a product may harbor a mutation but have the same migration rate as the wildtype product. To increase the sensitivity of the analysis we analyzed the products in two different types of gels. Of the forty cell lines, we were able to identify only one mutation/polymorphism in the cell line SK-LU-1. This nucleotide change did not result in a change of the amino acid sequence. It is possible that other mutations existing among the cell lines were not identified by PCR-SSCP, but it is more likely that MAP3K8 is not a frequently targeted gene for mutation in lung cancer.
The PCR amplifications from the SSCP analysis suggested that while mutation of the gene was uncommon, the different cell lines might express MAP3K8 at different levels. Some cell lines expressed very high levels compared to the 9HTE immortalized cell line and some cell lines appeared to have non-detectable PCR product visualized by standard PCR techniques. Based on these observations, it was possible that transcriptional and expressional aberrations instead of mutation of the gene may be more commonly associated with and contribute to lung tumorigenesis by MAP3K8. 141
Examination of the transcriptional expression of MAP3K8 through Realtime PCR of 28 lung cancer cell lines demonstrated that the expression varied greatly among the different cell lines. A 7600-fold difference separated the highest and lowest expression levels. Normal human bronchial epithelial cells were used as the established baseline value for expression, and these levels were validated when compared to the 9HTE non- transformed bronchial epithelial cell line that displayed similar levels. Overall, the
NSCLC cell lines appeared to increase expression of the mRNA, with three cell lines expressing more than 50-fold levels of RNA than the normal cells. The majority of SCLC cell lines harbored reduced expression of the mRNA. This suggested that there were differences between the two histologies of lung cancer and these expression characteristics may be significantly associated with lung tumorigenesis.
In order to support the RNA expression data, Western blot analysis of the protein levels was performed. The levels of protein expression did not correlate with the levels of
RNA expression in the cell lines. Interestingly, several cell lines displayed inversely proportionate levels. In addition, the cell lines also varied in the levels of a and b isoforms of MAP3K8 detected, although the SCLC cell lines with detectable levels of protein appeared to display both isoforms more frequently.
The lack of concordance between the mRNA and protein levels is not understood.
It is known that the MAP3K8 has a very short half-life associated with a non-ubiquitin proteolytic degradation of the protein (Gandara et al., 2003). The protein can be stabilized through binding to the NF-kB p105 protein. However, this binding also inactivates the protein. It is the p105-free MAP3K8 that is activated and able to activate the MAP kinase and other signal transduction pathways (Waterfield et al., 2003; Belich et al., 1999). To 142 date it is not known in what molecular context the MAP3K8 protein exists in the lung cancer cell lines examined. It is possible that the cell lines expressing high levels of mRNA but little to no protein may be constantly turning over a pool of active protein and constitutively activating the downstream pathways of MAP3K8 in a manner similar to mutational activation of the protein. Thus, increased expression of MAP3K8 may be a contributing factor towards tumorigenic transformation if the protein is constantly translated, activated, and subsequently degraded. Likewise, the high level of protein displayed in cell lines that had extremely low levels of transcript may be detectable because the proteins exist in a bound, inactivated state where the protein is not-degraded.
Both these scenarios are speculative at this point. And many experiments need to be conducted in order to examine the relationships among MAP3K8 transcription, translation, activation, and protein stabilization. What has been shown in lung cancer cell lines is that alterations of the RNA and protein levels of MAP3K8 exist. At this point, it remains to be shown what contributions these alterations have on tumorigenic development. Since MAP3K8 is infrequently mutated in lung cancer cells, it is unlikely that alterations of the gene are one of the primary steps determining tumorigenic progression as occurs with Ras mutation or loss of Rb. However, the altered transcription of the gene may be a contributing factor in the initiation and promotion of a normal cell to a tumor cell, especially if increased expression of the gene ultimately results in increased activation of growth and survival pathways such as NF-kB and MAPK. Increased activation of these pathways would provide a selective advantage to a cell that is progressing towards tumorigenic transformation. 143
One concern raised when using tumor cell lines for analysis of tumorigenic alterations is whether or not the cell lines are representative of the primary tumor cells.
Battacharjee et al. (2001) recently performed expression profiling of 186 primary lung tumors and 17 normal lung specimens using the Affymetrix DNA microarrays. MAP3K8 was one of the thousands of genes analyzed. A scatter plot of the different expression levels shows that the expression varies among the different tumors (Figure 1). The levels were calculated by subtracting the background expression from the foreground expression
(Expression Units). Since several tumors had higher background than foreground levels, these tumors were designated as 0 Expression Units for analysis. 144
Figure 1 (above). Expression level of MAP3K8 with the Affymetrix DNA microarray (Collected from Battacharjee et al., 2003). Scatter plot analysis of the Expression units for each of the 203 specimens analyzed. The different tumor types are separated by shape and color. Expression units below zero were listed at a value of zero for plotting and analysis. The specimen are listed in order are primary adenocarcinoma (Adeno), adenocarcnoma developed from a metastatic tumor cell (Ad-meta), squamous cell carcinoma (Squamous), carcinoid lung tumors (Carcinoid), small cell lung cancer (SCLC), and normal lung specimen (Normal).
The mean value of expression units (+/- SEM) was calculated for each of the tumor types and grouped as total non-small cell lung cancers (n=180), primary adenocarcinomas (n=127), metastatic adenocarcinomas (n=12), squamous cell carcinomas
(n=21), carcinoid tumors (n=20), small cell lung cancers (n=6), and normal lung specimens (n=17) (Figure 2). There were no large cell lung cancers analyzed. There was not a difference between the total NSCLCs, primary adenocarcinomas, squamous carcinomas, or carcinoid lung tumors and the normal lung expression. Interestingly the metastatic adenocarcinomas and the primary adenocarcinomas had different expression levels and the metastatic adenocarcinomas were also different from the normal cells, displaying a lower mean expression value. Squamous cell tumors displayed a much lower mean expression value than any of the tumor types and normal cells. Overall, there was not much difference between the mean expression of MAP3K8 in the tumor types and normal cells according to the microarray data. 145
Figure 2. Mean value of MAP3K8 expression in the different tumor types. The expression units from the Affymetrix DNA Array (Battacharjee et al., 2001) were grouped and calculated for mean +/- SEM values. The specimens are listed in order as the grouped non-small cell lung tumors (NSCLC), primary adenocarcinomas (AD), adenocarcinomas developed from a metastatic tumor cell (AD-META), squamous cell carcinomas (SQ), carcinoid lung tumors (CARC), small cell lung cancers (SCLC), and normal lung specimens (NORMAL).
The values displayed in Figures 1 and 2 do not appear to correlate well with the values observed in the Realtime expression data from the lung cancer cell lines. The
SCLCs had equivalent if not higher levels than the normal cells. While the overall levels of expression did not appear to be much different, several of the tumors did display relatively high levels of expression. It is possible that these tumors represent an outlying population of tumors that display increased levels of MAP3K8 transcription contributing to their growth and survival. 146
However, there are limitations to the data from the DNA microarrays that may also explain discrepancies between the Realtime data presented in this dissertation and the array data. First, the expression levels of MAP3K8 in the array data are extremely low compared to other genes on the array (data not shown). None of the lung tumors expressed MAP3K8 levels greater than 70 expression units, whereas these same tumors expressed other genes at levels in the thousands of expression units. Such differences can be due to factors such as the abundance of mRNA transcript or hybridization efficiency on the array. Many of the tumors displayed negative levels of expression for MAP3K8 due to higher background than foreground levels. This indicates that the sensitivity of the array to MAP3K8 expression is very low and that the values attributed to these tumors may not be accurately expressed. A second limitation is that the expression levels on the array are not normalized as was the Realtime data, adding to increased variability among the samples. Third, the tumors that were analyzed were not micro-dissected. Surgically removed lung tumors have a very high percentage of normal cells permeating throughout the tumor mass. The mRNA expressed in the normal cells causes the expression values of the tumors to appear closer to normal specimen values. These three points suggest that the expression characteristics of MAP3K8 from the microarray may not be representative of the actual expression exhibited in lung tumor cells. However, more research, such as
Realtime PCR of microdissected lung tissue, is necessary to support any conclusions from this microarray data.
The mutation and expression data served to characterize alterations of MAP3K8 that may occur during tumorigenesis to influence cellular growth and survival.
Ultimately, it is the functional role of MAP3K8 in the cell that is important in cellular 147 activities. Therefore, it was essential to transfect the gene into an immortalized lung cell line and examine the changes that occur in the cell.
Unexpectedly, the first change observed was a slowed proliferation rate of the cells transfected with either the wildtype MAP3K8 or the oncogenic lung mutant MAP3K8. It was interesting that the slowed proliferation rate of both the wildtype and mutant transfected cells followed identical growth curves. However, only the mutant appeared to slow proliferation through a G0/G1 arrest. Since neither form of the gene induced apoptosis, this was the first indication that the mutant gene may exert oncogenic activities through functional differences from the wildtype rather than just increased kinase activity.
9HTE cells transfected with wildtype and mutant MAP3K8 displayed a similar proliferation inhibition as the mouse embryonic fibroblast (MEF) cells transfected with the gene. However, MEF cells transfected with MAP3K8 change to accelerated proliferation upon the loss of the p16 tumor suppressor locus, Cdkn2a (Lund et al. 2003).
If such is the case in lung cells, it is possible that MAP3K8 may exert oncogenic properties in lung cancer only after the loss of tumor suppressor genes such as p16 or Rb.
One other interesting characteristic of the transfected cells was the expression of the translated MAP3K8 protein. The 9HTE transfected cell lines displayed both isoforms of the protein at high levels compared to the empty vector transfected cell lines. However, the tumor cell lines displayed variable expression of the a and b isoforms. Therefore, controlled regulation of MAP3K8 protein translation and stabilization may be characteristics of non-transformed lung cells that are lost in tumor cells during tumorigenic progression. 148
To understand the growth curve changes, we examined the cells at the level of both activated transcription factors and the phosphorylation of important proteins.
Surprisingly, known downstream transcription factors of MAP3K8, such as NF-kB and
CREB, were not activated while other transcription factors not previously linked with
MAP3K8, such as Brn-3 and MRE, were activated or in activated, respectively, under non-stimulated conditions. Thus, over-expression of MAP3K8 in lung cells has intrinsic activities and functions associated with cell cycle control and regulation.
The role of MAP3K8 in the Brn-3 pathway is one of extreme interest because Brn-
3 up-regulates transcription of the anti-apoptotic protein, Bcl-2. Up-regulation of Bcl-2 by Brn-3 only occurs upon loss of wildtype p53 (Buddhram-Mahadeo et al., 1999). This relationship suggests that MAP3K8 activity may be tumorigenic during the stages of tumorigenic progression after p53 is mutated or lost. It is interesting to note that the preliminary RT-PCR data suggested that the mutant, but not wildtype MAP3K8 gene, appeared to up-regulate Bcl-2 gene in 9HTE cells. The transcription factor array also identified the c-Myb protooncogene as differentially regulated by the wildtype versus the mutant MAP3K8 genes. The wildtype gene activated the transcription factor but cells harboring the mutant gene displayed decreased levels of the activated transcription factor, supporting the possibility that the two proteins have different functions.
The transcription factor array was complemented with a protein phosphorylation array. Again, many known substrates did not demonstrate increased phosphorylation upon MAP3K8 transfection. The reasons for this remain unclear. It could be that
MAP3K8 requires stimulation for phosphorylation of these proteins, suggesting that there are activators and/or inhibitors of MAP3K8 that regulate its function. The other 149 possibility is that the specific phosphorylation sites examined by the array are not the amino acids targeted by MAP3K8, but it is other amino acids in the protein that are phosphorylated by MAP3K8. If this is the case, the data from this array is extremely important and may help to explain how two upstream kinases can utilize the same downstream protein to elicit different responses. Understanding these site-specific phosphorylation events in the signaling proteins is a key step in understanding their role in cellular activities.
The phosphorylation array identified pathways in which MAP3K8 had not been previously known to be involved. One such pathway is the mTOR/4E-BP1/p70S6K pathway. Wildtype MAP3K8 inhibited phosphorylation of these proteins; a somewhat contradictory activity for a known kinase. It is known that AKT, a protein associated with
MAP3K8, is also an activator of the mTOR pathways (Kane et al. 2002; Nave et al.,
1999). To explain the decreased phosphorylation of these proteins, it is possible that
MAP3K8 sequesters AKT and prevents downstream activation of the mTOR pathway.
Thus, the decreased phosphorylation of these proteins would be a non-specific artifact of wildtype MAP3K8 transfection. Since the mTOR pathways are important in tumorigenesis, there may exist therapeutic benefits to understanding how this inhibition occurs. However, it is also possible that the decreased phosphorylation observed was, in fact, a functional activity of MAP3K8. Since supporting data for these relationships does not exist, more research is needed to understand the role of MAP3K8 in these pathways.
PKC a/b and AMPKa site-specific phosphorylation was also observed in the transfected cell lines. This is the first report of these proteins being involved in MAP3K8 pathways. It is unknown if they are direct substrates of the protein or are phosphorylated 150 as the result of other proteins in the MAP3K8 pathways. Again, more research is necessary to identify the relationships that exist among these different proteins, and how these relationships work to influence cellular growth and survival.
The data in this dissertation suggest that MAP3K8 is regulated at multiple levels in lung cancer cells and may influence cellular initiation, promotion, and transformation.
While mutational activation of the gene is a rare event in lung cancer, altered regulation of
MAP3K8 was observed to occur at transcriptional, translational, and possibly post- translation levels in lung cells. It is still unclear what modifications contribute to these alterations, but these data indicate distinct cellular contexts are necessary for MAP3K8 to have a positive influence on cellular transformation and tumorigenesis. The known activities of the protein indicate that increased expression and activity of MAP3K8 may play functional roles in induction of inflammatory responses associated with tumorigenesis. Likewise, this dissertation illustrated additional proteins MAP3K8 affected including the transcription factors Brn-3 and c-Myb, and the PKCa/b and the mTOR/4E-BP1/p70S6K proteins. These additional activities associate MAP3K8 with other pathways involved in tumorigenic promotion and suggest that altered regulation may be an important event in cellular promotion and lung tumorigenesis. 151
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Acknowledgements
I would like to thank James Cherry and Dr. Narayan Bhat at the Gene Expression Laboratory at SAIC Frederick for their help with the Realtime expression data; Ned Ramsay in the Laboratory of Genetics at the National Cancer Institute for his assistance with the Western Blots; Barbara Taylor at the Laboratory of Cellular Carcinogenesis and Tumor Promotion at the National Cancer Institute for her help with the FACS analysis and TUNEL assay; and Dr. Stuart Yuspa at the Laboratory of Cellular Carcinogenesis and Tumor Promotion at the National Cancer Institute for the Cancer Research Training Award and Fellowship and the opportunity to conduct research at the National Cancer Institute.
I would like to thank Dr. Marshall Anderson and Dr. Jonathan Wiest for the training and mentoring throughout this project.
I would like to extend a personal thanks to Dr. Brian “Brother B” Johnson for his friendship and help at both the University of Cincinnati and the National Cancer Institute.
Finally, I would like to extend great thanks to my family: my parents, Jim and Kathi; my brothers, Jay, Phil, and Keith; and my sisters, Emily and Lauren, for the years of understanding and support.