Abstract. Background: Little is known about the dual role of
RAS-association domain family 1 (RASSF1) gene at 3p21.3 in
neuroendocrine tumors (NET) of the lung. Materials and
Methods: Twenty typical carcinoids (TC), 11 atypical
carcinoids (ATC), 11 large cell neuroendocrine carcinomas
(LCNEC) and 16 small cell lung carcinomas (SCLC) were
analyzed for RASSF1 promoter methylation, mRNA and protein
expression, and loss of 3p21.3 locus. Results: Promoter 1 was
hypermethylated in NET but not in paired non-neoplastic lung
tissues nor in 20 control NSCLC, with the degree of
hypermethylation paralleling tumor grade. RASSF1 A/E
isoform mRNA but not protein expression was lost in most NET
compared to NSCLC or non-neoplastic tissues. The
relationship between methylation level and mRNA or protein
loss varied by NET type, with significant correlation for
decreasing RASSF1 A protein in ACT, and marginal correlation
for down-regulated RASSF 1 A/E mRNA in TC, this suggesting
a non linear regulation by methylation in NET. No promoter 2
methylation was detected in NET; however, up-regulation of its
RASSF1 C transcript emerged as an adverse prognostic factor
in the LCNEC/SCLC group. A correlation was found between
3p21.3 allelic loss and decrease of RASSF1 A/E mRNA
(p=0.023) and protein (p=0.043) expression in ATC,
suggesting that 3p21.3 allelic loss contributed to the loss of
gene expression. Conclusion: RASSF1 A/E is likely to act as a
tumor suppressor gene in most pulmonary NET, and RASSF1 C
as an oncogene in high-grade tumors.
RAS GTPases make up a superfamily of molecular
switches, which regulate diverse and complex cellular
functions, including proliferation, differentiation, motility
and apoptosis in response to various extracellular signals.
Beside well-known RAS effectors, such as RAF and PIK-3,
a new eight exon-spanning gene member has recently been
described, the RAS-association domain family 1 (RASSF1)
(also known as RASSF1A, NORE2A, 123F2, RDA32 and
REH3P21), which maps to chromosome 3p21.3 (1, 2) and
encodes at least eight different transcripts (RASSF1 A-H)
under control of two promoters (promoter 1 and promoter
2), and alternative splicing modalities (3, 4). Both promoter
1 and promoter 2 contain CpG-rich areas, and methylation
of these regions has been implicated as important for
regulating these promoters (3, 4).
RASSF1A and RASSF1C are the two most common
isoforms expressed in several normal tissues (5-7). Expression
of RASSF1A is regulated by promoter 1, while expression of
RASSF1C in under control of promoter 2. Isoforms RASSF1B,
RASSF1D and RASSF1E have been found predominantly in
hematopoietic, cardiac and pancreatic cells, respectively (8),
under the same promoter as the RASSF1A isoform (4). Data
on the prevalence and biological significance of RASSF1F,
RASSF1G and RASSF1H isoforms are still lacking.
In human cancer, regulatory mechanisms of RASSF1A
expression, the best known member of this protein family
Correspondence to: Giuseppe Pelosi, MD, MIAC, Dipartimento di
Patologia Diagnostica e Laboratorio, Istituto Nazionale dei Tumori,
Via G. Venezian, 1, 20133 Milano, Italy. Tel: +39 0223902260, Fax:
+39 0223902877, e-mail: email@example.com
Key Words: Carcinoma, carcinoid, small cell carcinoma, large cell
neuroendocrine carcinoma, methylation, immunofluorescence,
ANTICANCER RESEARCH 30: 4269-4282 (2010)
Dual Role of RASSF1 as a Tumor Suppressor and
an Oncogene in Neuroendocrine Tumors of the Lung
GIUSEPPE PELOSI1,2, CATERINA FUMAGALLI1, MAURIZIO TRUBIA3, ANGELICA SONZOGNI1,
NATASHA REKHTMAN4, PATRICK MAISONNEUVE5, DOMENICO GALETTA6, LORENZO SPAGGIARI2,6,
GIULIA VERONESI6, ALDO SCARPA7, GIORGIO MALPELI7and GIUSEPPE VIALE1,2
1Division of Pathology and Laboratory Medicine,
European Institute of Oncology and National Cancer Institute, Milan, Italy;
2Department of Medicine, Surgery and Dentistry, University of Milan School of Medicine, Milan, Italy;
3Department of Biotechnology and Biosciences, Milano-Bicocca University, Milan, Italy;
4Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA;
5Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy;
6Division of Thoracic Surgery, European Institute of Oncology, Milan, Italy;
7Institute of Pathology, University of Verona School of Medicine, Verona, Italy
acting as a tumor suppressor, most often include gene promoter
methylation and loss of heterozygosity (9, 10), leading to
impaired apoptosis (4), increased cell migration (11, 12) and
proliferation (13-15). In turn, the functions of RASSF1C
isoform are still debated, inasmuch as its role as either tumor
suppressor (16) or direct stimulator of cell proliferation (7, 17,
18) has been reported in both normal and several tumor tissues.
Neuroendocrine tumors (NET) of the lung constitute a
heterogeneous group of neoplasms including relatively
indolent lesions with longer life expectation (typical [TC]
and atypical [ATC] carcinoids) and very aggressive tumors
with dismal prognosis (large cell neuroendocrine carcinoma
[LCNEC] and small cell lung cancer [SCLC]) (19). Several
molecular prognostic factors have been proposed (20-24), but
histological typing still remains the most powerful predictor
of clinical course (25, 26). The greatest challenge for treating
these tumors, however, still remains the separation of patient
subsets with different tumor biological behavior within each
diagnostic category. Therefore, investigations on factors
implicated in the development and progression of these
tumors, as well as those affecting survival rates, are clearly
warranted, inasmuch as they could provide helpful
prognostic and predictive information.
Prior studies have shown that the RASSF1A isoform is
down-regulated by methylation in most SCLC and pulmonary
ATC in comparison with TC (1, 27-29). Epigenetic silencing
of RASSF1 A promoter by methylation and allelic loss of its
locus at 3p21.3 are also thought to be relevant to the
development of NET derived from the foregut (30-33),
abdominal paraganglioma (34) and adrenal glands (34, 35), as
well as in the growth of several types of pediatric malignant
tumors (36, 37). Furthermore, the presence of both RASSF1A
methylation and 3p21.3 allelic loss has been associated with
malignant behavior of foregut NET (30). In contrast, no SCLC
thus far examined has shown evidence for CpG island
methylation of RASSF1C (1, 5, 27), but a comprehensive
analysis of the methylation status, the prevalence and the
clinical implications of diverse RASSF1 isoforms across the
whole spectrum of pulmonary NET is still lacking.
In this study, we analyzed RASSF1 promoter methylation
and 3p21.3 allelic loss in relation to RASSF1A and RASSF1C
mRNA and protein expression in a series of 58 pulmonary
NET including TC and ATC, LCNEC and SCLC, as well as
20 NSCLC (10 squamous cell carcinomas and 10
adenocarcinomas) and non-tumor samples used as controls.
In addition, we studied the clinicopathological correlates of
these various parameters.
Materials and Methods
Strategy of study, selection of patients and adequacy of samples.
The RASSF1 gene generates at least five different transcripts
(isoforms A to E) under two promoters and alternative splicing
modalities. Since epigenetic silencing by methylation seems to play
a role in regulating this expression, we designed and conducted a
quantitative methylation analysis of both gene promoters by means
of a twofold approach including pyrosequencing technology (38-43)
and a customer-designed real-time methylation-specific polymerase
chain reaction (PCR) (QMSP) assay.
Using island size >100 bp, GC percentage >50, and
observed/expected ratio >0.6 as selection criteria and MethPrimer
software (44), two CG dinucleotide-rich regions were confirmed in
the promoter 1 sequence (GenBank accession number DQ444319;
island 1: 133 bp, start 424, end 556; island 2: 636 bp, start 564, end
1199) and one CG dinucleotide-rich region in the promoter 2
sequence (GenBank accession number NC_000003 from 3031 to
3770; 637 bp, start 48, end 684) (Figure 1), as previously reported
(9). Beta-actin (ACTB) was used as the internal reference
housekeeping gene in all experiments, inasmuch as it showed the
lowest standard deviation in nonneoplastic lung tissue samples after
comparison with other housekeeping genes (GAPDH, 18S, and beta-
Representative samples of both tumor tissue and paired
nonneoplastic lung parenchyma were included from 58
neuroendocrine tumor patients, comprising 20 TC, 11 ATC, 11
LCNEC and 16 SCLC, which were collected at the European
Institute of Oncology between 1997 and 2006. Ten adenocarcinoma
and ten squamous cell carcinoma samples collected at the same
Institution in the same time frame were used as a control group of
non-NET. All tumor patients entering the study had been
exhaustively studied with clinical history, physical examination,
respiratory function tests, radiological imaging and routine
laboratory profile prior to surgery, and all but three patients (2
bearing TC and 1 SCLC) underwent radical surgery inclusive of
extended mediastinal lymph node dissection (median value: 17
excised lymph nodes). There were 31 females and 27 males in the
patient cohort under investigation, with ages ranging from 30 to 80
years in the former (mean±SD: 59.6±11.8, median: 62 years) and
from 22 to 82 years in the latter (mean±SD: 60.1±12.6, median: 62
years). Complete follow-up information on relapse and survival was
available for all but two SCLC patients. Representative data on the
entire patient cohort according to the tumor histology is reported in
Table I. All patients gave informed consent to be enrolled into the
study that had been approved by the Institutional Review Board.
All nonneoplastic and tumor tissues had been immediately
removed at the time of surgery, and in part snap-frozen in liquid
nitrogen for DNA or RNA extraction within 10 min after excision,
in part fixed in formalin and embedded in paraffin for 6 to 8 hours
for routine histopathological examination. Diagnostic assessment
was carried out according to the criteria of the 2004 WHO
classification (19), and only tumor samples containing at least 80%
tumor cellularity were included in the study.
DNA and RNA extraction. Ten-μm-thick frozen sections were cut
from each tissue sample, either tumoral (containing at least 80% of
neoplastic cells) or nonneoplastic lung, and immediately processed
for DNA (NucleoSpin Tissue Kit; Machery-Nagel, Germany) or
RNA (RNeasy Mini Kit; Qiagen, Milan, Italy) extraction according
to the manufacturer’s instructions. RNase digestion was also
included into the DNA extraction protocol to obtain RNA-free
samples (Qiagen). Samples were then eluted in 100 μl of sterile
filtered water for DNA and in 50 μl of RNase-free water for RNA,
both being stored at –80˚C until use. The final yield of extracted
DNA and RNA was measured spectrophotometrically.
ANTICANCER RESEARCH 30: 4269-4282 (2010)
Pyrosequencing evaluation. Pyrosequencing analysis was carried out
in the current investigation (38-43) to thoroughly assess the
methylation status of both promoter 1 (island 1 and 2) and promoter
2. Island 2 of promoter 1 was arbitrarily split into pre-transcriptional
and post-transcriptional regions, as the latter spans encoding exon
1 of the relevant gene (Figure 1). In every tumor case, the results
were expressed as the median value of the methylation levels of all
CpG dinucleotides present in the relevant regions.
Quantitative (real-time) methylation-specific PCR (QMSP) assay. A
QMSP assay approach was carried out in the current investigation to
analyze island 2 methylation of the pre-translational sequence
according to many studies thus far published, which have pointed to
this region as being important for affecting protein expression (45-47).
Briefly, sodium-bisulfite treatment for DNA modification was
performed with EZ DNA Methylation Kit (Zymo Research, Orange,
CA, USA) according to the manufacturer’s instructions. Bisulfite-
modified DNA was then used as template for QMSP, designing
appropriate primers and probes for promoters 1 and 2 of RASSF1
gene as indicated in Table II. QMSP was carried out in a reaction
volume of 25 μl, each reaction mixture consisting of 600 nM of
each primer, 200 nM probe, TaqMan®universal PCR Master Mix
NO Amperase UNG 1× and 30 ng of modified DNA. Amplification
was performed in an ABI PRISM 7700 Sequence Detector (Applied
Biosystems, Foster City, CA, USA) using as thermal cycling
conditions denaturation steps at 95˚C for 10 min followed by 45
cycles at 95˚C for 15 s and 60˚C for 1 min. All samples were run in
triplicate and repeated again if they did not match with each other.
Each run of amplification included commercially available, CpG
dinucleotide-completely methylated DNA (CpGenome Universal
antibodies/ab/abhome) as a positive control, and three different
negative controls, either omitting DNA or using leukocyte DNA
from healthy volunteers, the latter either sodium bisulfite-
unmodified or sodium bisulfite-modified. Five serial dilutions from
50 to 1.563 ng/ml of the positive control were used for constructing
standard curves for each amplification run. Linear amplification
(with correlation coefficient, 0.999 to 0.995; slope, 3.25 to 3.35)
was obtained in each experiment and the ‘input amount’ values of
each sample were calculated by plotting Ctvalues on the
corresponding standard curve. To determine the relative levels of
methylated promoter DNA, the amount of the methylated gene of
interest was compared with the amount of DNA modified, based on
the internal reference gene, to obtain a ratio that was then multiplied
by 100 to give a percentage value (target gene/ACTB ×100).
DNA, Millipore at http://www.millipore.com/
Quantitative (real-time) PCR assay for RASSF1 isoforms. After
retro-transcription of RNA with MULV Reverse Transcriptase
Pelosi et al: RASSF1 in Neuroendocrine Lung Tumors
Figure 1. General diagram of RASSF1 promoter and the strategy used in the study for highlighting the methylation status in both promoter 1 and
promoter 2. As island 1 of the promoter 1 partially spans encoding exon 1 of the relevant gene, the CpG island has been arbitrarily divided into pre-
translational and post-translational sequences.
(Applied Biosystems) according to the manufacturer’s protocol to
obtain a final cDNA concentration of 30 ng/μl (thermal conditions
included 10-min random hexamer incubation step at 25˚C, followed
by a 48˚C extension for 30 min and 95˚C denaturation for 5 min),
real-time PCR evaluation was carried out with an ABI Prism 7700
Sequence Detector (Applied Biosystems) for both tumor and paired
nonneoplastic tissue samples. The amplification was achieved using
commercially available, intron-spanning TaqMan®Gene Expression
Assays (Applied Biosystems) for the diverse RASSF1 gene
transcripts (Hs00945257_m1 specific for isoform A and isoform E,
without distinguishing from each other [hence indicated as isoforms
A/E]; Hs00945679_m1 for isoform C; Hs00945252_m1 for isoform
D; Hs00945680_m1 for isoform F; and Hs00945677_m1 for
isoform G), as well as for β2-microglobulin (Hs99999907_m1) as
housekeeping target gene. No specific assay was available for
RASSF1B and therefore this transcript was not addressed in our
study. Thermal cycling conditions for all assays included 2 min at
50˚C, 10 min at 95˚C to activate the TaqMan®, followed by 40
cycles at 95˚C for 15 s and 60˚C for 1 min according to the
manufacturer’s instructions. All assays were repeated in triplicate,
averaging the results for each sample. The specificity of all PCR
reactions was checked by omitting cDNA from every assay as
negative controls and using commercially available normal lung
tissue RNA as positive controls (Human Lung Total RNA from
Applied Biosystems and MPV Total RNA Human Adult Lung from
Stratagene [Agilent, Santa Clara, CA, USA]).
The relative quantification of the target gene expression was
calculated by means of the comparative CTmethod (ΔΔCT)
β2microglobulin (CT REFERENCE) and as calibrator the mean of CT
values of the two commercial RNA samples (Human Lung Total
RNA and MPV Total RNA Human Adult Lung). Briefly, the ΔCT
value (ΔCT=CT RASSF1–CT REFERENCE) was calculated for each
sample under investigation, then compared with the calibrator
Biosystems), using as endogenous reference
ANTICANCER RESEARCH 30: 4269-4282 (2010)
Table I. Clinicopathological characteristics of 58 pateints with neuroendocrine tumors.
0.278 Positive LN %
*Presence of cases with incomplete information about pN status, vital status and recurrences; TC: typical carcinoids; ATC: atypical carcinoids;
LCNEC: large cell neuroendocrine carcinoma; SCLC: small cell lung carcinoma.
sample (ΔΔCT=Δ CT test sample–ΔCTcalibrator sample) to obtain a
2–ΔΔCTthreshold corresponding to the amounts of the relevant
mRNA normalized to an endogenous reference and relative to a
Immunofluorescence and fluorescence in situ hybridization (FISH)
assays. Frozen samples of both tumor and paired nonneoplastic lung
tissue were only immunostained for RASSF1A only because of the
lack of commercially available antibodies to other isoforms at the
time of the current investigation. Four-μm-thick air-dried sections
were fixed in acetone for 5 minutes, reacted with the mouse
monoclonal antibody 3F3 (IgG1) specifically recognizing the C1
domain of RASSF1A molecule (Abcam, Cambridge, UK), and then
incubated with a fluorescein isothiocyanate-conjugated goat anti-
mouse secondary antibody (Jackson ImmunoResearch, West Grove,
PA; USA), the former at a dilution of 10 μg/ml for 60 minutes, the
latter of 40 μg/ml for 30 min, both at room temperature. The
specificity of all immunoreactions was double-checked by
substituting the primary antibody with a non-related isotypic mouse
immunoglobulin at a comparable dilution, and with normal serum
alone. Tumors were considered immunoreactive for RASSF1A if
cell membrane, cytoplasmic or nuclear immunofluorescence was
observed. Immunoreactivity of nonneoplastic bronchial and alveolar
epithelia was used as internal positive controls in all cases.
Immunoreactivity on either tumor or nonneoplastic cells was
evaluated semiquantitatively and arbitrarily on a scale from negative
to 3+. Briefly, samples were considered negative if staining was
either completely absent or observed in fewer than 5% cells; 1+
cases showed fluorescence in 5-25% cells; 2+ cases in 26-50% cells
and 3+ cases exhibited immunoreactivity in the majority of cells (51
to 100%) (Figure 2).
FISH assay for RASSF1 gene was performed on 4-μm-thick,
consecutive paraffin-embedded sections using a home-made
SpectrumOrange-labeled DNA probe for the relevant gene or a
commercially available conjugated Cy3, SpectrumOrange-labeled,
centromeric enumeration probe (Abbott-Vysis, Downer Grove, IL,
USA). Briefly, BAC clones specific for RASSF1 (RP11-894C9),
belonging to the Roswell Park Cancer Institute libraries (Pieter J.
de Jong at http://bacpac.chori.org/), were selected according to
previous results from BAC library screening (data not shown).
Clones were validated by FISH analysis on normal fibroblasts to
confirm their expected chromosomal localization. Paraffin sections
were hybridized with the relevant probe labeled by nick translation
(48), using 500 ng of probe labeled with either Fluorolink Cy3-
dUTP or Fluor-X-dCTP (Amersham, Buckinghamshire, UK). At
least 60 nuclei were counted for RASSF1 signals and alpha-satellite
sequences of chromosome 3 centromere (CEP3) on consecutive
tissue sections. Results were expressed as the mean value of either
gene copy number (GCN) or RASSF1:CEP3 signal ratio per cell.
Monosomy for 3p21.3 locus containing RASSF1 gene was defined
by a mean of <1.5 signals per cell (49).
Statistics. Qualitative data are presented as frequencies and
percentages and compared using chi-square test, Fisher’s exact t-test
or Mantel-Haenszel test for trend as appropriate. Continuous data
were expressed using median values and contrasted employing the
Wilcoxon signed-rank test for pairs or the Kruskal-Wallis test if
medians were analyzed between two or more groups, respectively.
All correlation tests were performed using Spearman’s rank test (r).
Overall survival was defined as the time between surgery and the
last follow-up or cancer-related death. If a patient died without
cancer recurrence, the patient's survival time was censored at the
time of death. Only lung cancer-related deaths or recurrences were
considered as events. Disease-free survival was calculated from the
date of surgery to the date of progression or the date of last follow-
Pelosi et al: RASSF1 in Neuroendocrine Lung Tumors
Figure 2. Immunofluorecence results for RASSF1A antibody in tumor
samples showing 3+ staining in the majority of tumor cells (A), 1+
staining in 5-25% tumor cells (B), and negative staining, i.e. lack of
immunoreactive cells (C).
up. Survival estimates were calculated with Kaplan-Meier’s method
(50) and compared by the log rank test (51). Any statistical test was
considered significant if the corresponding p-value was ≤0.05.
Hypermethylation: Promoter 1 hypermethylation is specific to
NET and inversely correlates with tumor grade. Methylation
of RASSF1 promoter 1, either CpG island 1 or island 2, was
specific to NET, inasmuch as it was consistently lacking in
nonneoplastic lung tissue samples and NSCLC. The level of
promoter 1 methylation varied according to tumor grade, with
TC and ATC exhibiting lower levels of methylation than
LCNEC and SCLC, in both island 1 (p=0.032) and island 2
(pre-translation region, p=0.001; post-translation region
p<0.001) sequences, when analyzing the four groups of NET
together. In particular, higher levels of promoter 1 methylation
were seen in ATC as compared to TC, and SCLC as compared
to LCNEC using pyrosequencing and QMSP (Table III).
When considered as a whole and compared with NSCLC or
nonneoplastic lung tissue, NET showed higher levels of
methylation for both island 1 and island 2 of the promoter 1,
whereas no methylation was detected in the promoter 2 in any
NET, NSCLC or nonneoplastic tissue sample tested (Table
III). In all NET types there was a close correlation between
island 1 and island 2 methylation levels, independently of the
type of assay under evaluation (Table IV).
RASSF1 mRNA content: NET of the lung show decreased
levels of RASSSF1 A/E mRNA and increased levels of
RASSF1 C mRNA compared to non-tumor tissue and
NSCLC. We found that NET as a group showed down-
regulation of RASSF1A/E mRNA (under promoter 1) as
compared to nonneoplastic lung tissue samples, with 32 of
ANTICANCER RESEARCH 30: 4269-4282 (2010)
Table II. Primers used in the study for quantitative (real-time) methylation-specific PCR (QMSP) assay.
Gene Sequence (5’-3’) Length No. of
examined accession position
GenBank Amplicon Amplicon
TGG TGA TGG AGG AGG TTT AGT AAG T
AAC CAA TAA AAC CTA CTC CTC CCT TAA
6FAM ACC ACC ACC CAA CAC ACA ATA ACA AAC ACA TAMRA
GCG TTG AAG TCG GGG TTC
CCC GTA CTT CGC TAA CTT TAA ACG
6FAM ACA AAC GCG AAC CGA ACG AAA CCA TAMRA
GTT CGG GCG TAC GCG TAT A
CGC CCA TAA CCG TAC CCG
6FAM TAC GCG TAT ACG TAC GTA CGC GAT CG TAMRA
Y00474 390-522133 bp
PROMOTER 1 R
NC000003 3514-3624 111 bp
ACTB primers and probe were located in areas without CpG nucleotides, thus amplifying the modified DNA independently of the methylation status
of CpG nucleotides; F: forward; R: reverse.
Table III. Methylation of RASSF1 promoter 1 and promoter 2 in the tumor series under evaluation.
ATC p-Value LCNEC SCLC
(n=11) (TC vs. (n=11)
Island 1 methylation
Island 2 methylation
Pyrosequencing 35.147.50.045 40.2 63.50.061 46.213.0 <0.001 0.0
TC: Typical carcinoid; ATC: atypical carcinoid; LCNEC: large cell neuroendocrine carcinoma; SCLC: small cell lung carcinoma. QMSP: quantitative
methylation-specific protocol; NSCLC: non-small cell lung carcinoma samples. In the table, median values of percentage of promoter methylation
58 (55%) NET showing lower levels of methylation than
paired nonneoplastic controls. In contrast, RASSF1C mRNA
(under promoter 2) was overexpressed in 52 out of 58 (90%)
NET in comparison with paired nonneoplastic lung tissue
samples (RASSF1A/E vs. RASSF1C, p<0.001). Among NET,
RASSF1A/E mRNA was more conserved in carcinoids than
LCNEC/SCLC, whereas five cases of SCLC exhibited lower
levels of RASSF1C (Table V). No differences in the level of
RASSF1A/E and RASSF1C mRNA were found comparing
TC to ATC, and LCNEC to SCLC, apart from a significant
down-regulation of RASSF1A/E isoform in SCLC than
LCNEC (p=0.041) (Table VI).
When considering NET cases as a whole, fewer expressed
RASSF1A/E mRNA (55% of cases, p=0.0415) and more
RASSF1C mRNA (90% of cases, p<0.001) in comparison
with NSCLC, whether taking into account the tumor to
nonneoplastic lung tissue ratio (Table V) or the median
mRNA expression (Table VI). Differences in RASSF1A/E
mRNA levels were significantly different for each NET
tumor type (except LCNEC) compared to NSCLC.
No appreciable levels of RASSF1D, G and F isoforms (all
under promoter 1) were found in any NET, NSCLC or
nonneoplastic tissue sample under evaluation (data not shown).
Relationship between methylation status, RASSF1A/E mRNA
content and RASSF1 A protein expression. Correlation of
methylation levels with mRNA or protein expression yielded
variable results in the different tumor types under evaluation.
In NET, association of methylation (island 2, post-translational
region) and reduced RASSF1A/E mRNA content was detected
marginally in TC (r=–0.361, p=0.118) only, whereas no
correlation of methylation and reduced mRNA level was
identified in ATC, LCNEC, and SCLC (data not shown).
Neither was there any significant relationship found in
NSCLC between methylation and RASSF1A/E mRNA content.
Significant association for island 1 (r=–0.641, p=0.0337)
or marginal for island 2 (r=–0.479, p=0.136) methylation
and loss of RASSF1A protein expression was observed in
ATC but not other in types of NET. In NSCLC, there was a
marginal relationship between methylation and down-
regulated RASSF1A protein expression for both island 1
(r=–0.403, p=0.078) and island 2 (r=–0.409, p=0.073).
The proliferation index of NET did not correlate with
methylation status, RASSF1C or A/E mRNA content or
RASSF1A protein expression. No correlation was identified
between RASSF1A/E mRNA content and RASSF1A protein
expression in any tumor tissue, whereas an inverse relationship
was found in nonneoplastic lung tissue (r=–0.583, p=0.0601).
Clinical implications: Increased RASSF1C mRNA level
predicts recurrences and shorter survival in high-grade
NRET, independently of tumor stage. As LCNEC and SCLC
did not differ as to survival, we considered high-grade NET
as a single category for analysis (Figure 3). Carcinoid
tumors, either typical or atypical, cannot be separately
analyzed for survival or recurrence because of the low
number of events in this group.
We found that increased methylation of island 1 or island
2 was marginally associated with better overall survival in
the group of high-grade neuroendocrine tumors, with a
hazard ratio (HR) of 1.036 (95% CI: 0.995-1.079, p=0.088)
for island 1, and HR 1.040 (95% CI: 0.995-1.087, p=0.084)
for island 2 hypermethylation. Moreover, an increased
content of RASSF1C mRNA (median value 1.5) emerged as
dismal prognostic factor for overall survival (p=0.036) and,
marginally, for disease-free survival (p=0.112). (Figure 4)
Likewise, survival of these patient was strictly associated
with increased levels of RASSF1C mRNA, inasmuch as 6 out
of 7 patients who died of disease had tumor mRNA levels
>1.5 in comparison with 7 out of 18 of the surviving patients
(p=0.0392, test for trend). This association with poorer
prognosis was also maintained when the results were
stratified for regional lymph node-positive patients (pN1:
stage II; pN2: stage III) (Figure 5).
Univariate Cox’s survival analysis for continuous variables
confirmed the significant association between RASSF1C
mRNA content and overall (HR: 2.271; 95% CI: 1.281-4.027,
p=0.005) and disease-free survival (HR: 1.763; 95% CI:
1.107-2.807, p=0.0169). Methylation status, mRNA content
of isoforms other than RASSF1C, protein expression, Ki-67
labeling index and the other clinicopathological variables
listed in Table I were not associated with patients’ survival.
FISH analysis of RASSF1 gene. The Distribution of FISH
alterations for RASSF1 gene is presented in Table VII.
Monosomy was found in 57% of NET and 50% of NSCLC,
but in none of the nonneoplastic lung tissues. Likewise, there
were no significant differences in GCN among different
types of NET (data not shown).
A positive correlation was found between increased GCN
(FISH signals per cell) and RASSF1A/E mRNA (r=0.667;
p=0.023) or protein (r=0.721; p=0.043) expression in ATC,
suggesting a regulation by 3p21.3 loss in this tumor type.
Although similar results were found to some extent when
using pyrosequencing and QMSP (Table IV), functional
implications (see below) emerged only when exploiting the
former, probably because of the lower amounts of CpG
dinucleotides highlighted by QMSP in contrast with the
pyrosequencing strategy, which allows all known CpG
islands to be evaluated in a given genetic region. A key result
of our investigation is that that promoter 1 hypermethylation
of the RASSF1 gene controlling the expression of A, B, D
and E isoforms was limited to NET of the lung. Methylation
Pelosi et al: RASSF1 in Neuroendocrine Lung Tumors
ANTICANCER RESEARCH 30: 4269-4282 (2010)
Figure 3. Overall (A) and disease-free (B) survival curves according to tumor histological subtype.
Figure 5. Overall (A) and disease-free (B) survival curves of patients with large cell neuroendocrine carcinoma/small cell lung cancer and node-
positive disease according to RASSF1C mRNA expression.
Figure 4. Overall (A) and disease-free (B) survival curves of patients with large cell neuroendocrine carcinoma and those with small cell lung cancer
according to RASSF1C mRNA expression.
of this region was not seen in any samples of
adenocarcinoma or squamous cell carcinoma, and all the
paired nonneoplastic lung tissue samples exhibited lower
levels of methylation or even no methylation. This striking
difference supports the nonrandom role for this epigenetic
change in the development of pulmonary NET.
We found that the extent of methylation paralleled the
increasing grade of clinical aggressiveness of NET from TC
to ATC to LCNEC and SCLC, with lowest level of
methylation seen in TC and highest levels in SCLC. The
methylation levels of low/intermediate grade tumors (TC,
ATC) were significantly lower than those in high-grade
tumors (LCNEC, SCLC). These observations are in keeping
with previous studies on RASSF1 gene silencing in NET of
the lung, documenting increased levels of promoter 1
hypermethylation in most SCLC and ATC when compared to
pulmonary TC (1, 27-29).
We also found that the levels of RASSF1A/E mRNA,
which are under the control of promoter 1, were globally
down-regulated in the diverse categories of pulmonary NET
when compared with the corresponding samples of paired
nonneoplastic lung tissues or NSCLC, thereby establishing
a close relationship between gene promoter hypermethylation
and RASSF1 gene silencing, and confirming once again a
general function of the tumor suppressor gene for A/E
isoform in the development of NET of the lung. The level of
RASSF1A/E mRNA was more conserved in carcinoids than
LCNEC/SCLC and particularly low in SCLC, where it was
significantly lower than in LCNEC (p=0.041), further
supporting the functional role of tumor suppressor for
RASSF1A/E isoform in NET with a fine modulation
paralleling tumor grade.
Despite the striking hypermethylation of RASSF1
promoter 1 and the global decrease of RASSF1A/E mRNA
levels in NET, we found no correlation between methylation
and mRNA expression levels in individual NET (except for a
marginal association in TC only). A possible explanation for
this is that the quantitative extent of promoter methylation
(as reflected by the median value of methylation used
arbitrarily in our analysis) is not a significant determinant of
gene silencing, and even small levels of methylation
occurring in critical CpG islands may be sufficient to cause
loss of mRNA expression. In this context, if the relationship
of methylation and mRNA level is non-linear, it may be
interpreted as insignificant. Similar results were also found
in our series of NSCLC, thereby suggesting a non-linear
relationship of promoter methylation and relevant mRNA
expression when using a quantitative approach. Further
investigations on a larger series of NET and NSCLC and
different cut-off criteria are currently in progress in our
laboratory to address this important issue.
Similarly, RASSF1A protein level showed variable
correlation with methylation: Promoter 1 methylation was
associated with RASSF1A protein loss in ATC and to some
extent NSCLC, but not in any other NET. Furthermore, we
did not identify an association between RASSF1A mRNA and
protein level in any tumor type under evaluation. However,
an inverse correlation of mRNA and RASSF1A protein was
found in nonneoplastic lung tissue, supporting the belief that
post-translational modifications may contribute to the
RASSF1 A protein level regulation (37, 52). However, we
cannot completely exclude the possibility that our analysis of
RASSF1A protein levels by immunofluorescence may have
some technical limitations in terms of quantitative resolution,
and also, this reagent is relatively new and has not been
extensively thus far studied in lung cancer samples (53-56).
Despite these limitations, the association between methylation
and RASSF1A protein loss in ATC points to a function of the
recessive oncogene for this isoform and a role for island 1
that has not previously been reported in pulmonary NET.
Studies with human normal placenta reporting RASSF1A
protein loss in association with hypermethylation have
Pelosi et al: RASSF1 in Neuroendocrine Lung Tumors
Table IV. Relationship of RASSF1 promoter 1 methylation in neuroendocrine lung tumors.
Tumor type Number of casesPre-translation
TC: Typical carcinoid; ATC: atypical carcinoid; LCNEC: large cell neuroendocrine carcinoma; SCLC: small cell lung carcinoma; Island 1
pyrosequencing is used as reference for correlation tests; r value indicates correlation index according to the Spearman rank method.
explored island 2 (54), as have most of the published studies
on NET at different anatomical sites, with similar findings in
terms of hypermethylation across the diverse histologies (1,
5, 9, 27-37, 57). An inverse functional correlation with
corresponding mRNA isoform levels has been found for
island 2 hypermethylation in SCLC (1) and corresponding
tumor cell lines (1, 5), in human normal placenta (54), in
several malignant pediatric tumors and derived tumor cell
lines (36) and in pancreatic cancer cell lines (58), but not in
esophageal carcinoma (59). As discussed earlier, we did not
find a relationship between hypermethylation of either island
1 or island 2 and decrease in RASSF1A/E mRNA content in
the NET, aside from a marginal association in TC for island 2
(r=–0.361, p=0.118). These partially discrepant results could
be due to the different methods used for the assays in
different studies (1, 5).
In NET at different anatomical sites (30-33, 35) and in
many pediatric malignant tumors with neuroendocrine
differentiation (36, 37), epigenetic silencing of RASSF1
gene may also be caused by allelic loss of its locus at
3p21.3, a chromosomal region harboring several tumor
suppressor genes (52, 60). In our study, 57% of NET
exhibited fewer than 1.5 GCN per cell, indicative of
monosomy, whereas the remaining 43% comprised values
between 1.5 and 1.9 indicative of wider dispersion of losses
along chromosome 3p that variably includes RASSF1 gene.
These alterations were found to be independent of tumor
type, whether neuroendocrine or common lung carcinoma,
confirming the close relationship between 3p21.3 alterations
and lung epithelial cell carcinogenesis (52, 60).
Interestingly, increased FISH signals per cell correlated
positively with RASSF1A/E mRNA and protein expression
ANTICANCER RESEARCH 30: 4269-4282 (2010)
Table V. Distribution of RASSF1 isoform based on tumor to normal ratio according to the histological subtyping.
Tumor type No. of Isoforms A/E
p-Value Isoform C
cases T:N ratio ≤1T:N ratio >1T:N ratio ≤1 T:N ratio >1
TC: Typical carcinoid; ACT: atypical carcinoid; LCNEC: large cell neuroendocrine carcinoma; SCLC: small cell lung carcinoma; NET:
neuroendocrine tumors; NSCLC: non-small cell lung carcinoma.
Table VI. Analysis of RASSF1 mRNA in neuroendocrine lung tumors, NSCLC and nonneoplastic lung tissue.
RASSF1 A/E, ratio T:N
RASSF1 C, ratio T:N
All data are expressed as 2–ΔΔCTthreshold; median values were taken into account for the analysis; TC: typical carcinoid; ATC: atypical carcinoid;
LCNEC: large cell neuroendocrine carcinoma; SCLC: small cell lung carcinoma; NSCLC: non-small cell lung carcinoma.
Table VII. Comparison of RASSF1 FISH analysis of neuroendocrine tumors (NET), NSCLC and nonneoplastic lung tissue.
VariableGCN <1.5 (%) GCN >1.5 (%)
p-ValueMedian GCN (range)RASSF1:CEP3 ratio (range)
Nonneoplastic lung tissue (n=58)
0.94 (0.78-1.2) <0.001
GCN: Gene copy number; NSCLC: non-small cell lung carcinoma.
in ATC, suggesting that in addition to regulation by
methylation, RASS1A/E mRNA and RASSF1 A protein
expression may also be regulated by 3p21.3 allelic loss in
ATC. This is in accordance with a two-hit mechanism of
RASSF1 inactivation whereby either methylation or 3p21.3
loss are involved in silencing of RASSF1 gene expression in
NET of the lung. A role for RASSF1A isoform silencing by
gene promoter hypermethylation and/or allelic loss of its
locus at 3p21.3 (9, 10) has also been invoked for the
development (30-33) and progression (30, 31, 33) of most
foregut-derived NET, sporadic and MEN2-associated
pheochromocytoma and abdominal paraganglioma (34, 35),
and several types of pediatric malignant neoplasm, including
rhabdomyosarcoma, medulloblastoma, retinoblastoma and
neuroblastoma (36, 37). In all these tumor types, RASSF1A
isoform is likely to serve as a tumor suppressor gene,
leading to impaired apoptosis (4), increased cell migration
(11, 12) and increased proliferation rate (13-15). This
observation is not surprising because 3p21.3 is known to
harbor a tumor suppressor gene cluster whose alterations
have been found in most types of human cancer (52, 60).
The differential distribution of RASSF1 gene promoter 1
hypermethylation across the whole spectrum of pulmonary
NET we documented in our investigation probably reflects a
specific change contributing to the development of these
tumors rather than a simple epiphenomenon associated with
pulmonary epithelial cell carcinogenesis, inasmuch as it was
less demonstrable in common types of lung carcinomas. A
practical application of this finding could be a diagnostic
one, for example when experiencing difficulties in
differentiating low- to intermediate-grade carcinoids from
high-grade neuroendocrine carcinomas, which require
completely different therapeutic approaches, in the event of
severe crush artifacts in small biopsy samples (61). In
addition, distinction of basaloid squamous cell carcinoma
from SCLC can present a diagnostic challenge with standard
morphological approaches. Analysis of RASSF1 promoter
methylation could service as a useful ancillary tool in this
Our investigation showed that promoter 2 was never
hypermethylated in NET, NSCLC and paired nonneoplastic
lung tissue samples, in accordance with previously reported
data indicating that none of the SCLC samples thus far
examined showed evidence of CpG island methylation of
RASSF1C (1, 5, 27). Accordingly, increased levels of
RASSF1C isoform mRNA were found in all types of lung
carcinomas we examined in comparison with nonneoplastic
tissue samples, in keeping with the hypothesis that RASSF1C
isoform could affect lung carcinoma development according
to a function of the dominant oncogene, independently of the
occurrence of a neuroendocrine differentiation. Interestingly,
this dual functions have been described in other genes too,
such as retinoblastoma – a prototypical tumor suppressor
gene, which in some settings can act as an oncogene by
blocking cell differentiation, as has been described for
erythroid differentiation in cultured mouse erythroleukemia
cell lines (62).
Another original aspect of our investigation regards the up-
regulation of RASSF1C transcript as an adverse prognostic
factor in the group of high-grade NET, including LCNEC and
SCLC, for both overall (HR: 2.271; p=0.005) and disease-
free survival (HR: 1.763; p=0.0169), independently of tumor
stage. These findings are in accordance with a function of the
dominant oncogene for RASSF1C favoring the progression
and impairing the survival of patients with pulmonary high-
grade neuroendocrine carcinomas as previously reported in
other tumor tissues (7, 17, 18). Increased hypermethylation
of the island 1 or island 2 was also marginally associated with
overall survival in the group of patients with high-grade NET,
with HR 1.036 (95% CI: 0.995-1.079, p=0.088) for the
former and HR 1.040 (95% CI: 0.995-1.087, p=0.084) for the
latter, suggesting a role of a tumor suppressor gene for
RASSF1A/E isoforms as previously indicated for SCLC (5).
In conclusion, our study supports the view that the
RASSF1 gene plays an important role in the development and
progression of pulmonary NET, with dual function as tumor
suppressor gene in all types of NET for RASSF1A/E isoform
and as oncogene impairing patients’ survival for RASSF1C
isoform in high-grade NET.
This work was supported by Associazione Italiana per la Ricerca sul
Cancro and is dedicated to the memory of Carlotta, an
extraordinarily lively girl who untimely died of cancer in the prime
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Received July 4, 2010
Revised September 1, 2010
Accepted September 7, 2010
Pelosi et al: RASSF1 in Neuroendocrine Lung Tumors