Lung cancer is the leading cause of cancer mortality in both men and women in the United States 1. Non-small cell lung carcinomas arise from the respiratory epithelium and progress through well-defined pathological stages prior to becoming invasive and metastatic tumors. While many studies have identified lung tumor markers of clinical or prognostic significance, survival rates for this deadly disease have remained essentially unchanged for the past 30 years. The slow advance in treating lung cancer is due in part to continued gaps in our understanding of the molecular mechanisms of lung tumorigenesis. Thus, studies that aid in our understanding of molecular mechanisms of lung tumorigenesis are important steps towards developing better detection, prevention and treatment of this disease.

Evasion of apoptosis by tumor cells is a critical step during tumorigenesis. The serine/threonine kinase Akt is a critical mediator of anti-apoptotic signaling in eukaryotic cells and is activated in a signaling cascade downstream of Ras activation and phosphoinositide-3-kinase (PI3K) 2. Amplification of PI3K is common in many tumor types, including lung cancer 345 and in lung cancer is correlated with increased phosphorylation of Akt 4. Activation of Akt, as measured by phosphorylation of the protein, is also increased in multiple tumor types including lung cancer 678910. Increased phosphorylation of Akt kinase has also been reported in developing bronchial hyperplasias and dysplasias 71112 and pre-neoplastic atypical alveolar hyperplasia 13, indicating that activation of this pro-survival pathway may be a relatively early event in lung tumorigenesis.

The NF-κB transcription factor family can stimulate both pro- and anti-apoptotic signals. Many studies have described a critical role for NF-κB activity in promoting cell survival. Increased staining for NF-κB subunits has been detected in breast 14 and cervical carcinoma 15. Inhibition of NF-κB activity either pharmacologically or genetically can sensitize tumor cells to pro-apoptotic agents 16171819 or to tumor necrosis factor-α (TNF-α) induced apoptosis 20. Proteasome inhibition, which blocks the degradation of inhibitor of κB (IκB) protein, thus blocking NF-κB nuclear translocation and activation, also sensitized NSCLC cells to apoptosis 2122. Similarly, expression of a super-repressor form of IκBα sensitized lung cancer cell lines to apoptosis-inducing drugs 1623. The super-repressor form of IκBα, as well as a dominant negative form of IKK, also blocked Ras-mediated transformation of cells 2425 and expression of the IκB super-repressor inhibited anchorage independent growth and metastatic spread of human lung cancer cell lines in a tumor xenograft model 26. Furthermore, Akt can activate the transcriptional potential of the p65/RELA subunit of NF-κB 2728, providing a potential link between Akt kinase activity and NF-κB activation. Western blot analysis has demonstrated over expression of the p50 subunit of NF-κB in lung cancer 29, but localization of NF-κB family members has not been described in lung tumors. Nevertheless, abundant evidence links NF-κB transcriptional activation with lung tumorigenesis.

Several NF-κB-regulated genes that function in control of apoptosis have been described including cIAP-1, cIAP-2, A1/Bfl1, Traf1, Traf2 and Bcl-X_L 30313233. cIAP-1 and -2 are members of the baculoviral IAP repeat-containing (BIRC) gene family. cIAP-2/BIRC3 is expressed in lung adenocarcinoma cell lines 34 and can be induced by TNF-α 35. Elevated expression of cIAP-2/BIRC3 has been reported in human NSCLC 363738 and elevated expression of the related proteins XIAP/BIRC4 and survivin/BIRC5 are also seen in NSCLC 3639, implicating the BIRC family of proteins as important mediators of lung tumorigenesis.

While the expression of pro-survival genes has been examined in some detail in lung tumors, relatively few studies have examined the expression of these proteins in pre-neoplastic lesions. To determine the presence and abundance of pro-survival proteins in developing lung neoplasia, we examined human bronchial biopsies of various grades (normal, hyperplasia, mild, moderate and severe dysplasia, carcinoma in situ and carcinoma) for the presence of phospho-Akt, p65/RELA and cIAP-2/BIRC3 and determined their localization within cells. The results obtained provide new insight into the distribution of pro-survival genes in human lung neoplasia and pre-neoplasia. This information may be useful in designing studies to test the importance of these pathways experimentally and ultimately may lead to improved diagnostic or therapeutic strategies for human lung cancer.

The pathways utilized by lung tumors to evade apoptosis, allowing the tumors continued survival and growth, are incompletely characterized. Equally important are the pathways that become activated in IEN lesions before they become invasive cancer. In this study, we examined the presence and cellular localization of the phosphorylated form of Akt kinase, the transcription factor p65/RELA and the cellular inhibitor of apoptosis protein cIAP-2/BIRC3 in human bronchial IEN lesions. In normal human bronchial epithelium, staining for phosphorylated Akt was present as a faint cytoplasmic stain. Staining intensity increased with increasing pathology grade and was present in a nuclear, perinuclear or plasma membrane pattern. The intensity of p65/RELA staining also increased with increasing pathology grade. Furthermore, more biopsies had nuclear localization of p65/RELA with advancing pathology grade, indicating nuclear translocation of the protein and the potential for transcriptional activation. The apoptosis inhibitor cIAP-2/BIRC3 also had increased staining intensity with increasing pathology grade. These results indicate that Akt, p65/RELA and cIAP-2/BIRC3, components of an anti-apoptotic pathway, are elevated in pre-neoplastic lung lesions.

The serine/threonine kinase Akt is a central mediator of anti-apoptotic pathways in eukaryotic cells 2. Activation of this kinase occurs when it is itself activated by PI3K-dependent protein kinase 1 or 2 (PDK1 or 2). Previous studies have noted an increase in the amount of phosphorylated Akt in a number of different human malignancies including cancer of the lung 4712, head and neck 46, prostate 9, and in multiple myeloma 847. In addition, loss of the tumor suppressor phosphatase and tensin homologue (PTEN), a lipid phosphatase that inhibits PI3K activity, is found in a subset of lung tumors 484950. Increased staining for phosphorylated Akt was also frequently observed in human bronchial pre-neoplastic lesions 4712. In bronchial IEN, increased staining for phospho-Akt correlated with increased staining for phospho-FKHR, a transcription factor that directs expression of anti-apoptotic genes in response to Akt signaling 7. Our present results agree with previous studies demonstrating an increase in phospho-Akt staining in pre-neoplastic human bronchial lesions. We observed increased staining for the phosphorylated form of Akt beginning as early as mild dysplasia lesions in human bronchial biopsies.

The staining for phospho-Akt observed in the biopsies in our study was diverse, consisting of cytoplasmic, nuclear or plasma membrane associated patterns. As PDK must associate with PI3K at the plasma membrane to be activated, localization of Akt has often also been localized to the cytoplasm or plasma membrane 89. However, several targets of Akt are nuclear transcription factors and nuclear localization of Akt has been described in several other human tumor types 71247. The differences in subcellular localization observed for phospho-Akt in different biopsies may reflect the large number of pathways regulated by this kinase and the different mechanisms by which Akt can mediate its anti-apoptotic effects. The functional significance of the variable localization of phospho-Akt in human lung tumors remains to be determined.

The NF-κB transcription factor family is known to direct both apoptotic and anti-apoptotic signaling in eukaryotic cells 51. In unstimulated cells, NF-κB transcription complexes are held in the cytoplasm by IκB proteins, thus preventing their function as transcriptional activators in the nucleus. Cytokine or growth factor stimulation initiates a signaling cascade that leads to activation of the IκB kinase (IKK) complex that in turn phosphorylates IκB and targets it for ubiquitination and ultimately degradation by the proteasome. Degradation of IκB allows NF-κB to be translocated to the nucleus where it can activate transcription of target genes. Inhibition of NF-κB blocked Ras-mediated transformation of cell lines 24 indicating NF-κB activity is critical for Ras-mediated transformation. The Akt kinase has been reported to activate NF-κB by phosphorylation of IKK 52, causing degradation of IκB and nuclear translocation of NF-κB complexes, and by directly phosphorylating the p65/RELA subunit of NF-κB and increasing its transcriptional activity 2728. It should also be noted that Akt can be activated by NF-κB 53, suggesting the regulatory pathways utilized by these molecules is complex.

While ample evidence has implicated NF-κB activation as playing an important role in cell transformation, particularly in vitro, there is little data on the presence or localization of NF-κB components in human lung tumors. Chemotherapeutic agents induced NF-κB activity in NSCLC cell lines, increasing the cells resistance to these agents 17 suggesting that in addition to a role in tumor cell evasion of apoptosis, NF-κB may render lung tumor cells more resistant to chemotherapeutic agents. The NF-κB subunit p50 was increased in NSCLC as detected by immunoblotting but no information on localization of this protein within tumors was obtained 29. To our knowledge, no other study has examined the localization of the p65 subunit of NF-κB in lung IEN lesions or NSCLC. We therefore decided to examine human IEN lesions and lung carcinomas for the presence and localization of the p65/RELA subunit of NF-κB.

In our study, nuclear translocation of the p65/RELA subunit of NF-κB was significantly increased in moderate dysplasias compared to lower grade lesions. In the carcinomas, there was a reduction of p65/RELA nuclear positivity similar to the decreased intensity of phospho-Akt staining observed in carcinomas compared to moderate and severe dysplasias. The percentage of cells with positive nuclear staining in carcinomas was also very low compared to moderate and severe dysplasias generally being restricted to isolated foci. In addition to the increased number of biopsies with nuclear staining at higher pathology grades, the overall intensity of cytoplasmic tended to be increased in moderate dysplasias in both the epithelium and stroma compared to normal epithelium (although this difference was not statistically significant in our analysis). In a number of carcinomas, there was intense cytoplasmic staining for p65/RELA with an apparent paucity of staining in the nucleus. While these biopsies were scored negative for nuclear staining of p65/RELA, it was impossible to discount the possibility that nuclear p65/RELA was present in these cells but at levels lower than those observed in the cytoplasm. In the lung adenocarcinoma cell line NCI-H441 we have also observed higher levels of p65 immunoreactivity in the cytoplasm than the nucleus although these cells have very high levels of NF-κB activity as measured by reporter gene assays (JWT and MWA, unpublished data). It should also be noted that activation of NF-κB has been reported in lung cancer cells without an increase in nuclear localization 2454. In all lesions with positive nuclear staining, a majority of tumor cells did not contain nuclear p65/RELA staining. The focal nature of nuclear positivity may reflect the induction of this pathway in a subset of cells that has acquired new genetic or epigenetic changes that activate NF-κB and thus may activate downstream anti-apoptotic pathways.

Tumor cells acquire the ability to escape apoptosis that is normally initiated in damaged cells. One mechanism for inhibiting apoptosis is to block the activity of the effector caspases that initiate apoptosis by degrading specific cellular targets 55. The cellular inhibitor of apoptosis (cIAP) family of proteins, also known as the baculoviral IAP repeat containing (BIRC) family, can inhibit apoptosis by binding effector caspases and blocking their proteolytic activity. Several family members have been described including cIAP-1/BIRC2, cIAP-2/BIRC3, XIAP/BIRC4, survivin/BIRC5 and NAIP/BIRC1, and are thought to be important in tumorigenesis 45. We chose to examine the levels of cIAP-2/BIRC3 in developing lung neoplasia as it is expressed in lung tissue 38 and is a target for NF-κB transcriptional activation 31.

While increased staining for cIAP-2/BIRC3 in human lung adenocarcinomas has been reported 38, levels of cIAP-2/BIRC3 in IEN lesions in lung epithelium have not been reported. Staining for cIAP-2/BIRC3 was increased in the severe dysplasia/CIS category and in carcinomas compared to less advanced pathology grades. The increase in cIAP-2/BIRC3 staining occurred at a higher pathology grade than the grade at which phospho-Akt staining increased (mild/moderate dysplasias) or p65/RELA nuclear localization was increased (moderate dysplasia). This may indicate that inhibition of apoptosis via this protein is not required until later in lung tumorigenesis. While high levels of cIAP-2/BIRC3 expression correlated with nuclear localization of p65/RELA in some biopsies, this was not a consistent observation. The lack of consistent p65/RELA and cIAP-2/BIRC co-expression, in combination with the observation that the overall increase in cIAP-2/BIRC staining occurred at a later pathological stage than p65/RELA nuclear translocation, may indicate that cIAP-2/BIRC3 is not a direct target of p65/RELA in bronchial pre-neoplasia. The possibility remains that p65/RELA transcriptional activation initiates a program that may indirectly lead to cIAP-2/BIRC3 induction later in the progression of pre-neoplastic bronchial lesions.

While nuclear staining localization of p65/RELA and increased staining for cIAP-2 did not correlate, we observed a positive correlation between staining intensity for cIAP-2 and phospho-Akt. While it is impossible to state whether this indicates that cIAP-2 is regulated by activation of Akt phosphorylation, this is a possibility. It should be noted, however, that staining intensity for phospho-Akt occurred one pathology grade prior to the observed increase in cIAP-2 staining intensity.

Increased staining for phospho-Akt and cIAP-2/BIRC3 is present in pre-neoplastic human bronchial biopsy lesions compared to normal human bronchial epithelium. Additionally, the percentage of biopsies containing nuclear p65/RELA staining is increased in pre-neoplastic lesions compared to normal bronchial epithelium. The activation of genes involved in protecting cells from apoptosis in pre-neoplastic lesions suggests that the ability of lung epithelial cells to evade apoptosis is acquired prior to the cells becoming fully transformed. As pre-neoplastic lesions have not acquired all the genetic and epigenetic changes present in lung carcinoma, they may be more amenable to treatment with chemopreventive agents. Thus, identification of the pathways activated in human bronchial pre-neoplastic lesions is important in increasing our understanding of how these lesions develop into lung cancer with the eventual goal of targeting these pathways therapeutically.

The author(s) declare that they have no competing interests.

JWT reviewed histological staining, participated in design of the study and drafted the manuscript. YZ carried out the immunohistochemical analyses. JCL determined pathology grades of bronchial IEN lesions and reviewed histological staining. PWB determined pathology grades of lung carcinomas and helped draft the manuscript. SL participated in the design of the study, obtained IEN lesions and helped draft the manuscript. MWA conceived of the study, participated in study design and helped draft the manuscript. All authors read and approved the final manuscript.