| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Department of Medicine [P. P. M., P. M. T., S. M. J. R., P. Y., D. P. C.], Biostatistics Shared Resource [Y. S.], The Vanderbilt Ingram Cancer Center, Molecular Profiling and Data Mining Shared Resource [M. E. E.], and Departments of Pathology [M. E. E., A. L. G.], Biochemistry [M. D. W., J. A. P.], and Surgery [J. R. R.], The Vanderbilt Ingram Comprehensive Cancer Center, Vanderbilt University, School of Medicine, Nashville Veterans Affairs Medical Center [P. P. M.], Nashville, Tennessee 37232-6838
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Np63
. Western analyses revealed Np63 expression in normal bronchus and squamous carcinomas but not in normal lung or in adenocarcinomas. Furthermore, p63genomic amplification and protein staining intensity associated with better survival. We found a significant increase in CN in preinvasive lesions graded severe dysplasia or higher. Our data demonstrate that there is early and frequent genomic amplification of p63 in the development of squamous carcinoma of the lung and that patients with NSCLC showing amplification and overexpression of p63 have prolonged survival. These observations suggest that p63 genomic amplification has an early role in lung tumorigenesis and deserves additional evaluation as a biomarker for lung cancer progression. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and responsible for phosphatidylinositol 3'-kinase activity, maps to this region (3q26). We have shown that increase in PIK3CA gene CN correlates with increased protein kinase B activity, implicating the phosphatidylinositol 3'-kinase pathway in lung tumorigenesis. This amplicon also contains a number of other important genes, including p63, hTR, neutral endopeptidase, and somatostatin.
The p63 genomic sequence maps to the peak of the 3q amplicon (chromosome 3q27). p63 is a transcription factor that transactivates p53 target genes (3)
and induces apoptosis when expressed in cells (4)
. Although p63 was recently discovered, it is the most ancient member of the p53 family (5)
. p63is a p53 homologue that encodes six splice variants. Those splice variants possessing a p53-like NH2-terminal transactivating domain are known as TAp63 and have properties similar to p53 (3)
. In contrast, splice variants lacking the NH2-terminal transactivating domain, known as Np63, are thought to be inactive and function as dominant negatives, promoting growth and survival by competing for p53 binding sites (3)
. Interest in p63 stems from this "two genes in one" concept with agonist and antagonist properties that may be involved in tumor development. The Np63 splice variant is known to be the most commonly expressed isoform in squamous epithelia (3)
and is often found in head and neck and lung tumors with amplification of p63 (6)
.
The function of p63-specific splice variants in normal and malignant epithelial cells is subject to active investigation. In contrast to p53, loss-of-function mutations of the p63 gene are rarely found in human tumors (7)
. Some evidence suggests that Np63 may function as an oncogene. Np63 has been shown to inhibit transcription when transiently transfected into a p53 luciferase reporter assay (3)
. Overexpression of Np63 in Rat 1a cells increased tumor size in mice (6)
. Np63 was found to cause accumulation and signaling of ß-catenin, additionally supporting the oncogenic function of p63 (8)
. In addition, Np63 overexpression in keratinocytes may have a dominant negative effect on the endogenous p53 transcriptional activity required for UV-B-induced apoptosis in vivo (9)
. Finally, Np63 can function as a potent transcriptional repressor and dissociates from promoter binding sites of key growth inhibitory genes (p21 and 14-3-3
) during normal human keratinocyte differentiation (10)
. For these reasons, p63 gene amplification and overexpression may have important implications in tumorigenesis. The prevalence of p63 amplification in NSCLCs is unknown. Moreover, whether amplification correlates with overexpression of p63 and whether either abnormality has any role as a biomarker for the early diagnosis or for the prognosis of lung cancer has not yet been determined.
This study addressed the following questions: (a) what is the prevalence of p63 amplification in NSCLC? (b) is amplification associated with p63 protein expression, and if so, which p63 splice variant is preferentially expressed? (c) at what stage of lung tumor development is p63 amplified? (d) what is the prognostic value of p63 CN or protein expression in NSCLCs? and (e) are there significant relationships between p63 CN, p63 expression, p53 expression, and proliferation marker Ki-67 expression profiles?
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
NSCLC Tissue Microarrays.
Tissue microarrays of NSCLC were prepared from paraffin blocks and an equal number of normal lung tissues (11)
. There were 94 squamous carcinomas, 93 adenocarcinomas (including 16 bronchioalveolar carcinomas), and 30 large cell carcinomas represented on the tissue microarrays. Archived tissue blocks from 1989 to 2002 were retrieved from the files of Vanderbilt University and the Nashville Veterans Affairs Medical Center pathology departments. For all tissue blocks, H&E-stained sections were reviewed by our pathologist (A. L. G.). Areas to be punched for array production were carefully marked. All 217 tumors were punched in triplicate, normal samples only once. Cores 0.6 mm in diameter were taken from the selected area of each specimen and inserted into a recipient paraffin block. Five-µm sections were cut from the arrays and mounted onto charged slides. Every fifteenth section was stained by H&E to confirm the presence of the histological feature of interest (tumor or normal histology).
FISH.
BAC clones for specific genes were selected from different libraries: p63 (RP03B2743); FHIT (2175D15); and centromeric chromosome 3 (CEP3) probe was purchased from (Vysis, Inc., Downers Grove, IL). Dual color FISH was performed on interphase nuclei in tissue sections as described earlier (2)
. Briefly, 2 µg of BAC DNA were labeled by nick-translation with either digoxigenin-dUTP or Cy3-dUTP. Labeled probes were separated from the reaction using spin columns and denatured. Tissue sections were deparaffinized and treated with sodium thiocyanate for 10 min followed by pepsin (4 mg/ml) digestion in 0.2 N HCl for 10 min at 37°C. Slides were then denatured in 70% formamide/2x SSC for 5 min at 72°C and incubated with a hybridization mixture consisting of 50% formamide, 2x SSC, Cot-1 DNA, and 100 ng of both digoxigenin-labeled and Cy3-labeled BAC DNAs. After 2 nights of incubation at 37°C, the slides were washed and counterstained with antifade solution containing 4',6-diamidino-2-phenylindole. Test and reference hybridization signals were scored in 50 nuclei for each tumor core under a 100x immersion objective. Nuclei in which the nuclear boundaries were broken were excluded from the analysis. For invasive tumors, the average number of spots counted in 50 nuclei was reported as CN for the test gene. To address the interpretation of genomic amplification of the p63gene in a subset of 85 NSCLCs, we performed FISH and reported p63/centromeric chromosome 3 CN ratio. For preinvasive lesions, we took advantage of chromosome 3p deletion in a percentage of preinvasive lesions and used the FHIT locus on the opposite arm of chromosome 3 to report the ratio of the mean count for the test probe to the mean count of the reference probe on the opposite chromosome arm. We determined the reliability of the assay by testing interobserver variability in FISH spot counting. Two observers were asked to count fluorescent spots in 50 nuclei for both test (red) and control genes (green) for 10 separate tumor biopsies represented on a tissue microarray (observers blinded to histological subtype). We found an excellent inter-observer correlation with r2 of 0.64 (r = 0.8032, P < 0.0001).
RT-PCR.
Total RNA was isolated from tissue specimens using the TRIzol protocol (Invitrogen, Carlsbad, CA). Reverse transcription reactions were carried out for 1 h at 42°C–44°C and contained 1 µg of total RNA, 250 ng of oligo(dT), 1x deoxynucleotide triphosphate mix, RNase inhibitor (Promega), 1x RT buffer, and 200 units of SuperScript II RT (Invitrogen) in a total volume of 20 µl. Amplification of TAp63 and Np63 splice variants was performed in 50 µl of reaction mixture consisting of sense and antisense primers for TAp63, Np63, and Np63
; 2 µg of cDNA, 1x deoxynucleotide triphosphate mix, 1x PCR buffer, 1.5 mM MgCl2, and 2.5 units of AmpliTaq Gold DNA Polymerase (Perkin-Elmer, Wellesley, MA). PCR conditions were: 3 min at 95°C; followed by 30 cycles of 94°C for 30 s; 54°C for 1 min; and 70°C for 1 min, with a final extension at 70°C for 5 min. The following primers were used: TAp63, 5'-ATGTCCCAGAGCACACAG-3'(sense) and 5'-AGCTCATGGTTGGGGCAC-3' (antisense); Np63, 5'-TCTGGACTATTTCACGACCC-3' (sense) and 5'-TCACTCCCCCTCCTCTTTG-3' (antisense); and Np63, 5'-TACAGGCAACAGCAACAGCA (sense) and 5'-CTATGGGTACACTGATCGG-3' (antisense).
Western Blotting.
Surgically resected specimens of squamous cell carcinomas and adenocarcinomas of the lung, as well as adjacent histologically normal tissue, were obtained from the Vanderbilt University Medical Center. Samples were snap-frozen and stored at -80°C until protein lysates were prepared. Specimens were homogenized and lysed in buffer containing 50 mM HEPES, 250 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1% NP40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 tablet of protease inhibitors (Roche, Indianapolis, IN)/10 ml of buffer. Lysates were centrifuged at 14,000 K for 15 min at 4°C, and the supernatant was removed. Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA), and 100 µg of total protein from each sample were separated on a 10% SDS-polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, United Kingdom) at 100 V for 1 h at room temperature. Blocking was performed in 5% milk + 1X Tris-Buffered Saline/0.1% Tween-20 (TBST) overnight at 4°C. Primary antibody against p63 (4A4; Oncogene Research Products, San Diego, CA) was diluted 1:500 in PBS. Secondary antibody was horseradish peroxidase-conjugated mouse antihuman immunoglobulin (Promega, Madison, WI) diluted 1:2500 in PBS. Detection was done by enhanced chemiluminescence (Pierce, Rockford, IL). Equal loading was confirmed by Ponceau Red staining and by probing the blot with antibody against ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA).
IHC.
IHC staining was performed by the avidin-biotin complex method using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) as described previously. Slides were deparaffinized in xylene and hydrated through a graded alcohol series before being placed in 3% H2O2-PBS blocking solution for 5 min to inhibit endogenous peroxidase activity. Slides were then digested with 0.01% trypsin in PBS at 37°C for 15 min, followed by rinsing with plain PBS for 5 min at room temperature. Antigen unmasking was done in 10 mM citrate buffer (pH 6.0) for 10 min, and slides were then allowed to cool at room temperature for 30 min. Blocking serum consisted of 50 µl of normal horse serum in 5 ml of PBS and was applied for 30 min at room temperature, followed by washing in PBS. Slides were incubated with either p63 4A4 primary antibody (Oncogene Research Products) diluted 1:500 in PBS, p53 DO-7 antibody diluted 1:100 (Dako, Carpinteria, CA), or Ki-67 MIB-1 antibody diluted 1:50 (Immunotech, Marseilles, France) for 1 h at 4°C, washed in PBS, and treated with the corresponding biotinylated secondary antibody for 30 min at room temperature. After washing with PBS, sections were exposed to the ABC Elite reagent for 5–10 min at room temperature. Reactions were developed with 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and counterstained with hematoxylin. In invasive carcinomas, p63 and p53 staining was scored 0–4 on intensity. In preinvasive lesions, p63 was scored based as follows: 0 for no staining; 1 for basal layer; 2 for basal and parabasal layer staining; and 3 for full thickness staining. Ki-67-labeling index was defined as the percentage of nuclei immunohistochemically positive for MIB-1 among a minimum of 500 cancer nuclei (12)
. One of the main concerns about tissue microarrays is that the 0.6-mm biopsies may not represent the whole tumor specimen because of tumor heterogeneity. To address this question, we arrayed triplicates of tumors as described previously (13)
. We determined intercore variation by assessing variability for three biomarkers (p53, p63, and Ki-67) across triplicates tested by IHC and found low coefficients of variation (coefficient of variation, SD/mean) 0.34, 0.22, and 0.21, respectively, attesting for the low level of variability while sampling the same tumor multiple times.
Survival Analysis.
CN ratios (continuous variable) and IHC scores (parametric variables) were tested for correlations and for survival analysis. The average scores of triplicate biopsies were used for FISH analysis. Maximal immunostain scores from triplicates were used for IHC and data obtained from the tumor registry allowed survival analysis. Clinical data elements were obtained from the Bioinformatics Core of the Vanderbilt Ingram Cancer Center. Data analysis included Spearman correlation coefficients and Kaplan-Meier survival estimates with Cox proportional hazards regression models. Survival analysis was calculated from date of diagnosis to date of death or last date of contact for those alive at the time of the analysis. Curves were compared by the log-rank test. All analyses were carried out with SAS statistical software (SAS Institute, Inc., Cary, NC).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The level of amplification showed internal consistency between the three punches taken from different regions of the same tumor. Among 10 tumors each punched in triplicates, the mean coefficient of variation for p63 CN was 32.38 ± 4.5 (standard deviation) while the coefficient of variation for FHIT CN was 37.5 ± 4.09. We also found a high correlation between p63 amplification and squamous histological subtype (r = 0.65, P < 0.001), yet the degree of p63amplification was independent of stage and grade. Specifically, CN values varied from 3 to 11.5 copies (average for 50 nuclei). Although the CN values for p63 varied significantly between cells, every cell layer was affected. None of the nontumor cells showed 3q amplification. To determine whether p63 genomic amplification was attributable to locus amplification or aneusomy, we determined CN for p63 and for centromeric chromosome 3 by dual color FISH in a subset of 85 NSCLCs. We found that increased CN was due to aneusomy of chromosome 3 in 21% of the tumors.
|
Np63, Np63, and TAp63 transcripts by RT-PCR in 24 NSCLCs, in 12 normal lung tissues, and in normal bronchial epithelium. Np63 transcripts were found in bronchial epithelium and in squamous carcinomas but not in normal lung or in adenocarcinomas (Fig. 2A)
. Np63 transcripts were expressed in normal bronchial epithelium and in some squamous carcinomas but not in adenocarcinoma or in normal lung (Fig. 2, B and C)
. TAp63 transcripts were found in striated skeletal muscle, bronchial epithelium, and some squamous carcinomas at low levels but not in adenocarcinomas or normal alveolar tissue (Fig. 2, B and C)
.
|
70,000 protein in squamous carcinomas (Fig. 3 A, B)
that was entirely absent in normal (Fig. 3 A)
tissue and in adenocarcinomas (Fig. 3 A, C)
. This migration is similar to that previously reported for Np63 (3
, 10) . The data shown in Fig. 3
are representative examples of 12 squamous, 12 adenocarcinomas and 12 matched normal lung tissue samples.
|
. In normal airway epithelium (Fig. 4A)
, p63 staining was nuclear and restricted to the basal cell layer. Alveolar epithelial cells did not stain with p63 (Fig. 4B)
. p63 expression was consistently found at high levels in squamous carcinoma (77 of 94) throughout the entire thickness of the tumor (Fig. 4E)
. The staining intensity was reduced within areas of additional differentiation and areas most distant from the vascular bed (Fig. 4E)
. p63 staining was present in only 17 of 93 adenocarcinomas (Fig. 4F)
and in 7 of 30 large cell carcinomas. Staining intensity for p63 could be directly correlated with p63 amplification in NSCLCs overall (Spearman correlation coefficient, r = 0.502, P < 0.0001); however, this correlation did not hold for squamous carcinomas (r = 0.04, P = 0.717). This inconsistency points to an unknown mechanism of p63 gene expression that is independent of genomic amplification.
|
|
). Severe dysplasia and CIS are almost invariably amplified for p63. Amplification of p63 correlated with p63 immunostaining (P < 0.001). In preinvasive tumors, p63 staining involved not only the basal but also the supra-basal layers and followed a pattern consistent with severity of histological grade (Figs. 4, C and D
, and 5B
). In invasive tumors, p63 was seen throughout the whole sheet of invasive tumor. All invasive tumors synchronous to high-grade preinvasive lesions exhibiting p63 CN amplification also showed p63 amplification. When invasive tumors did not show p63 amplification, their matched preinvasive tumors were also negative.
|
. This observation suggests that although p63 does not have a direct role in cell cycle regulation, p63 may allow a specific subpopulation of the epithelium to undergo either differentiation or proliferation given a specific signal.
|
3) exhibited a survival advantage versus the overall population (P < 0.05) and also among patients with squamous carcinomas (P < 0.05; Fig. 7A
). We also found a strong association between the intensity of p63 staining in squamous carcinomas and increased odds of surviving (P < 0.0174; Fig. 7B
). After adjusting for stage, patients with tumors displaying increased p63 CN still had prolonged survival. Histological grade did not affect survival.
|
3) in 30% of the tumors (Fig. 8)
. We found that Ki-67-labeling index had no prognostic value (P = 0.2327) in the overall set of tumors or in any of the histological subgroups. Ki-67 index did correlate with p63 CN (P = 0.002) but not with IHC (P = 0.5347). These data suggest that p63 is not a direct marker of proliferation and has a distinct role from p53 in tumor progression.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Np63, Np63, and TA splice variants are expressed in NSCLCs, and Np63 is the predominant splice variant expressed. In squamous cell carcinoma, p63 amplification and staining intensity are associated with better survival independently from stage and degree of differentiation of the tumor. We did not find a correlation between p63 immunostaining and p53 or Ki-67 expression. Chromosome 3q26-ter amplification, which includes the p63gene locus, is one of the most prevalent genomic abnormalities in solid tumors and is likely to play a critical role in tumorigenesis. Amplification of chromosome 3q has been described in squamous epithelial transformation from the lung (6) , head and neck (19 , 20) , esophagus (21) , bladder (22) , cervix (23 , 24) , and stomach (25) . We have previously observed that the size of the amplicon varies tremendously between tumors (15–70 Mb) and contains >300–600 known and unknown genes (2) . The relevance of most of these genes to tumor progression remains to be determined. We recently demonstrated that the presence of 3q amplification alone allows the distinction between squamous and adenocarcinoma in >75% of cases (2) . In the present study, we confirmed this in an independent tumor set and extended it to a larger number of NSCLCs. Both increased p63 CN and p63 staining intensity were strongly associated with squamous histological subtype. Although p63 was amplified in 11% of adenocarcinomas, half of these tumors were aneuploid and some exhibited deletion of one p63allele. Finally, 42% of large cell carcinomas also exhibited p63 amplification, yet these tumors are poorly differentiated and may actually represent poorly differentiated squamous carcinoma.
Although p63 CN did correlate with p63 staining intensity in NSCLCs overall, it did not correlate among 94 squamous cell carcinomas (P = 0.717). These observations are in agreement with data in head and neck cancer showing no correlation between genomic amplification and gene expression by comparative genomic amplification using quantitative RT-PCR in 10 tumors and 18 normal tissues (26) . Absence of correlation between gene amplification and protein expression in squamous carcinoma suggests that the degree of amplification does not regulate protein expression directly. The functional repercussions of p63amplification remain the subject of evaluation in the context of the large chromosome 3q amplicon.
p63 amplification occurs early in the development of lung cancer and may have important implications in early detection strategies. To evaluate CN in preinvasive lesions, we chose to determine CN ratio by dual color FISH, taking advantage of the frequent 3p deletion on 3p14 in NSCLC. This CN ratio (p63/FHIT) increases the sensitivity of our assay to detect small genomic abnormalities in tissue sections. FHIT is deleted in some NSCLCs (27, 28, 29, 30) , which we confirmed in up to 30% of squamous carcinomas.
p63 immunostaining data shows progressive increase throughout the depth of the epithelium from metaplasia to severe dysplasia and relates to the pathology. These findings, obtained in 43 preinvasive lesions, confirm data previously reported by Pelosi et al. (31) and Sniezek et al. (32) in a small series of head and neck and lung tumors. In particular, it is apparent that p63 expression increases progressively from preinvasive to invasive lesions during the transformation of squamous epithelia. Moreover, p63 immunoreactivity has been shown to be inversely correlated with both squamous cell maturation and nonsquamous differentiation in cervical intraepithelial neoplasia (24) . This progressive increase of p63 expression according to pathological grade makes p63 less attractive to early detection efforts and does not offer a major advantage to pathological distinction. In contrast, amplification of p63 is characteristic of high-grade (severe dysplasia and CIS) preinvasive and invasive squamous lung cancers but not earlier. In our set of 43 preinvasive lesions, 13 were found adjacent to invasive squamous carcinomas. For every high-grade preinvasive lesion where p63was amplified, the corresponding invasive tumor was also amplified. Low-grade preinvasive lesions found in the vicinity of invasive tumors did not show p63 CN gain regardless of the degree of amplification of the invasive tumor. This suggests that p63 amplification occurs during tumorigenesis when the preinvasive lesions commit to invasion and may therefore be a good biomarker for early detection of patients with or at risk for lung cancer. Given the quantifiable nature of p63 CN by FISH and to address obvious limitations of a retrospective study, we are planning a prospective evaluation of 3q amplification as a predictive biomarker for lung cancer development in biopsy specimens and in sputum from patients at risk for or with lung cancer. Because not all high-grade lesions develop into invasive tumors (33) , the management of preinvasive lesions is still a subject of controversy. Our study suggests that special consideration should be given to preinvasive lesions with p63 amplification. Furthermore, identification of these lesions may assist in monitoring the response to new chemopreventive agents.
The mechanism by which p63 amplification and p63 overexpression participate in tumor progression remains unclear. We confirmed that p63 is expressed at the basal layer of the airway epithelium, a layer that has regenerative potential (3
, 6
, 31)
. Because specific antibodies to the Np63 splice variant are not currently available, we could not confirm the predominant expression of this splice variant in preinvasive and invasive lung tumors by immunochemistry. Our data on localization of p63 and Ki-67 demonstrates a different pattern of expression suggesting that Np63 may not play a direct role in cell cycle regulation. At some concentration, Np63 may be toxic and cause cells to die (4)
. Once the cells have committed to proliferation, p63 may become less important functionally and may potentially be toxic. This may account for the reduced expression seen in more differentiated cancer cells with persistent genomic amplification.
We found that p63 amplification and overexpression are extremely prevalent in squamous carcinomas and that they are also associated with better survival. In a recent study, Pelosi et al. (31) examined the percentage of p63-positive cells by IHC, yet did not find an association between p63 expression and survival. Nevertheless, we found that the majority of cancer cells (>80%) stained for p63 in squamous carcinomas, and therefore, we tested whether the staining intensity of the cells rather than the percentage might correlate with outcome. Our data demonstrate a prolonged survival in patients with p63 gene amplification and a lower risk of dying for patients with tumors that have strong p63 immunostaining. We speculate that the reason for a survival advantage could involve the following: p63 functions as a survival factor that promotes transformation in the appropriate unstable genetic background. Once transformed, cells develop and keep p63 genomic amplification. As the cells further differentiate, p63 expression is decreased and may then be an important marker of cell differentiation and confer a survival advantage.
Because of sequence homology with p53, it has been suggested that p63 may have a similar pattern of expression in tumors. Our observation is that p53 and p63 overexpression occurs simultaneously in only 30% of squamous carcinomas of the lung and that there is no relationship between the two markers among the three histological subtypes studied. We also could find no correlation between p53 and p63 immunostaining for any stage or grade of differentiation. These results suggest independent roles for p63 and p53 during tumorigenesis. The functional interpretation of p53 and of p63 assessed by immunostaining remains a limitation of this study. There is no data available correlating p63 immunostaining and activity in human tumors. Similarly for p53, although mutated p53 generally shows strong immunoreactivity (17) , mutation of the gene can lead to overexpression of the protein or complete loss of expression (truncated protein; Ref. 34 ), making the immunostaining an imperfect predictor of p53 mutation status.
This genomic amplification of p63 in preinvasive lesions is also likely to be part of the same amplicon found in invasive squamous carcinomas. This genomic region of amplification on chromosome 3q26-3q28 also contains a number of other important genes, some of which have been called potential candidate oncogenes or are thought to be involved in tumor progression (PIK3CA, somatostatin gene, telomerase RNA component gene, and neutral endopeptidase gene). The specific role of these other candidate biomakers in tumor progression and their functional interactions remain to be determined.
Finally, p63 amplification allows us to identify two different classes of NSCLCs. Upon transformation, the majority of tumors developing from the bronchial epithelium acquire 3q amplification. This pattern suggests a different mechanism of tumor development from those of other cell types. Although all NSCLCs are currently treated identically, genes in the chromosome 3q amplicon may open a window for identification of potential targets for molecular intervention in preinvasive and invasive lung cancer. Taken together, our data suggest that p63 amplification and overexpression of Np63 are critical steps in the early development of NSCLC and that p63 amplification in particular may prove to be an excellent biomarker of squamous carcinoma progression.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 P. P. M. was supported, in part, by a research grant from the American Lung Association, the Vanderbilt Physician Scientists Program through the Vanderbilt Specialized Programs of Research Excellence in Lung Cancer Grant 5P50 CA 90949-02, and by the Office of Research and Development, Department of Veterans Affairs. 
2 To whom requests for reprints should be addressed, at The Vanderbilt-Ingram Comprehensive Cancer Center, 2200 Pierce Avenue, PRB 640, Nashville, TN 37212-6838. E-mail: pierre.massion{at}vanderbilt.edu
3 The abbreviations used are: PIK3CA, phosphatidylinositol-3 kinase catalytic subunit; CN, copy number; FISH, fluorescence in situ hybridization; FHIT, fragile histidine triad; NSCLC, non-small cell lung cancer; RT-PCR, reverse transcription-PCR; IHC, immunohistochemistry; CIS, carcinoma in situ.
Received 4/ 2/03. Revised 8/ 7/03. Accepted 8/26/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and Np63 can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes. Oncogene, 20: 3193-3205, 2001.[Medline]
Np63 induces ß-catenin nuclear accumulation and signaling. Cancer Cell, 1: 369-379, 2002.[Medline]
and 
Np63 in benign and malignant oral epithelial lesions. Int. J. Cancer, 87: 368-372, 2000.[Medline]
in normal and hyperdifferentiated mucosa of the upper aerodigestive tract. Otolaryngol. Head Neck Surg., 126: 598-601, 2002.[Medline]
This article has been cited by other articles:
|
|
 |
|
  S. Ocak, P. Chaurand, and P. P. Massion Mass Spectrometry-based Proteomic Profiling of Lung Cancer Proceedings of the ATS, April 15, 2009; 6(2): 159 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Qian, Y. Zou, J. S.M. Rahman, B. Lu, and P. P. Massion Synergy between phosphatidylinositol 3-kinase/Akt pathway and Bcl-xL in the control of apoptosis in adenocarcinoma cells of the lung Mol. Cancer Ther., January 1, 2009; 8(1): 101 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ogawa, R. Okuyama, T. Egawa, H. Nagoshi, M. Obinata, H. Tagami, S. Ikawa, and S. Aiba p63/p51-induced Onset of Keratinocyte Differentiation via the c-Jun N-terminal Kinase Pathway Is Counteracted by Keratinocyte Growth Factor J. Biol. Chem., December 5, 2008; 283(49): 34241 - 34249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. V. Pedchenko, A. L. Gonzalez, D. Wang, R. N. DuBois, and P. P. Massion Peroxisome Proliferator-Activated Receptor {beta}/{delta} Expression and Activation in Lung Cancer Am. J. Respir. Cell Mol. Biol., December 1, 2008; 39(6): 689 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. P. Massion, Y. Zou, H. Chen, A. Jiang, P. Coulson, C. I. Amos, X. Wu, I. Wistuba, Q. Wei, Y. Shyr, et al. Smoking-related Genomic Signatures in Non-Small Cell Lung Cancer Am. J. Respir. Crit. Care Med., December 1, 2008; 178(11): 1164 - 1172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Rosenbluth and J. A. Pietenpol The jury is in: p73 is a tumor suppressor after all Genes & Dev., October 1, 2008; 22(19): 2591 - 2595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-K. Chu, P.-M. Dai, H.-L. Li, and J.-K. Chen Transcriptional Activity of the {Delta}Np63 Promoter Is Regulated by STAT3 J. Biol. Chem., March 21, 2008; 283(12): 7328 - 7337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Marchini, M. Marabese, E. Marrazzo, P. Mariani, D. Cattaneo, R. Fossati, A. Compagnoni, R. Fruscio, A. A. Lissoni, and M. Broggini {Delta}Np63 expression is associated with poor survival in ovarian cancer Ann. Onc., March 1, 2008; 19(3): 501 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R Malaguarnera, V Vella, R Vigneri, and F Frasca p53 family proteins in thyroid cancer Endocr. Relat. Cancer, March 1, 2007; 14(1): 43 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. DeYoung, C. M. Johannessen, C.-O. Leong, W. Faquin, J. W. Rocco, and L. W. Ellisen Tumor-Specific p73 Up-regulation Mediates p63 Dependence in Squamous Cell Carcinoma Cancer Res., October 1, 2006; 66(19): 9362 - 9368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Barbieri, L. J. Tang, K. A. Brown, and J. A. Pietenpol Loss of p63 Leads to Increased Cell Migration and Up-regulation of Genes Involved in Invasion and Metastasis. Cancer Res., August 1, 2006; 66(15): 7589 - 7597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. M. Keyes, H. Vogel, M. I. Koster, X. Guo, Y. Qi, K. M. Petherbridge, D. R. Roop, A. Bradley, and A. A. Mills p63 heterozygous mutant mice are not prone to spontaneous or chemically induced tumors PNAS, May 30, 2006; 103(22): 8435 - 8440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. S. Helton, J. Zhu, and X. Chen The Unique NH2-terminally Deleted ({Delta}N) Residues, the PXXP Motif, and the PPXY Motif Are Required for the Transcriptional Activity of the {Delta}N Variant of p63 J. Biol. Chem., February 3, 2006; 281(5): 2533 - 2542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J.C. Slebos, Y. Yi, K. Ely, J. Carter, A. Evjen, X. Zhang, Y. Shyr, B. M. Murphy, A. J. Cmelak, B. B. Burkey, et al. Gene Expression Differences Associated with Human Papillomavirus Status in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., February 1, 2006; 12(3): 701 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.E. King, R.M. Ponnamperuma, M.J. Gerdes, T. Tokino, T. Yamashita, C.C. Baker, and W.C. Weinberg Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis Carcinogenesis, January 1, 2006; 27(1): 53 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Mills p53: link to the past, bridge to the future Genes & Dev., September 15, 2005; 19(18): 2091 - 2099. [Full Text] [PDF]
|
|
|
|
|
|
G. Tonon, K.-K. Wong, G. Maulik, C. Brennan, B. Feng, Y. Zhang, D. B. Khatry, A. Protopopov, M. J. You, A. J. Aguirre, et al. High-resolution genomic profiles of human lung cancer PNAS, July 5, 2005; 102(27): 9625 - 9630. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N Haruki, K S Kawaguchi, S Eichenberger, P P Massion, A Gonzalez, A F Gazdar, J D Minna, D P Carbone, and T P Dang Cloned fusion product from a rare t(15;19)(q13.2;p13.1) inhibit S phase in vitro J. Med. Genet., July 1, 2005; 42(7): 558 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Haruki, K. S. Kawaguchi, S. Eichenberger, P. P. Massion, S. Olson, A. Gonzalez, D. P. Carbone, and T. P. Dang Dominant-Negative Notch3 Receptor Inhibits Mitogen-Activated Protein Kinase Pathway and the Growth of Human Lung Cancers Cancer Res., May 1, 2005; 65(9): 3555 - 3561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. TONON, C. BRENNAN, A. PROTOPOPOV, G. MAULIK, B. FENG, Y. ZHANG, D.B. KHATRY, M.J. YOU, A.J. AGUIRRE, E.S. MARTIN, et al. Common and Contrasting Genomic Profiles among the Major Human Lung Cancer Subtypes Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 11 - 24. [Abstract] [PDF]
|
|
|
|
|
|
S.-i. Kurata, T. Okuyama, M. Osada, T. Watanabe, Y. Tomimori, S. Sato, A. Iwai, T. Tsuji, Y. Ikawa, and I. Katoh p51/p63 Controls Subunit {alpha}3 of the Major Epidermis Integrin Anchoring the Stem Cells to the Niche J. Biol. Chem., November 26, 2004; 279(48): 50069 - 50077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. P. Massion, P. M. Taflan, Y. Shyr, S. M. J. Rahman, P. Yildiz, B. Shakthour, M. E. Edgerton, M. Ninan, J. J. Andersen, and A. L. Gonzalez Early Involvement of the Phosphatidylinositol 3-Kinase/Akt Pathway in Lung Cancer Progression Am. J. Respir. Crit. Care Med., November 15, 2004; 170(10): 1088 - 1094. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kim, J. Han, J. Kim, and C. Park Maspin Expression Is Transactivated by p63 and Is Critical for the Modulation of Lung Cancer Progression Cancer Res., October 1, 2004; 64(19): 6900 - 6905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Daniely, G. Liao, D. Dixon, R. I. Linnoila, A. Lori, S. H. Randell, M. Oren, and A. M. Jetten Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium Am J Physiol Cell Physiol, July 1, 2004; 287(1): C171 - C181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Westfall and J. A. Pietenpol p63: molecular complexity in development and cancer Carcinogenesis, June 1, 2004; 25(6): 857 - 864. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
2 and strong when