Abstract. Background: Malignant pleural mesothelioma is a
highly aggressive cancer, with low overall survival. The
pathogenesis of mesothelioma is poorly understood. The aim of
this study was to identify potential genes overexpressed in
mesothelioma. Materials and Methods: A cDNA microarray was
used to identify potential genes that are activated in mesothelioma
cell lines. Overexpression of stathmin, a cytosolic protein that
regulates microtubule dynamics, was found. RT-PCR, Western
blot, and immunohistochemistry were used to confirm
overexpression in both cell lines and tumor samples. Results:
Using RT-PCR and Western blot, stathmin overexpression was
confirmed in seven mesothelioma cell lines. Increased stathmin
protein expression was also found in seven out of eight
mesothelioma tumor samples. Finally, stathmin expression in a
mesothelioma tumor was confirmed by immunohistochemistry.
Conclusion: For the first time, stathmin was shown to be
overexpressed in malignant mesothelioma. The overexpression of
stathmin in mesothelioma may offer a potential therapeutic target
and further studies are warranted.
Malignant pleural mesothelioma is an aggressive cancer which
arises from the pleural lining of the lung (1). Approximately
3,000 patients are diagnosed with mesothelioma annually in
the United States and the incidence is expected to increase (2-
4). Most patients present at a relatively late stage and have a
poor prognosis. Curative resection is usually not possible and
medical therapies have had limited benefit. The median
survival time is 12 months (5, 6).
Targeted therapies may improve the survival for patients
with mesothelioma, but the pathogenesis of mesothelioma
is poorly understood. Asbestos exposure is linked to the
majority of cases, but its exact role in oncogenesis is still
unclear (7, 8). Unlike many other epithelial cancers, the
activation of ras genes and inactivation of Rb and p53 genes
do not seem to be necessary for the development of
mesothelioma (9, 10). Alterations of several molecular
pathways, including epidermal growth factor receptor, cell
cycle regulatory genes, and developmental pathways have
been linked to mesothelioma (11, 12). There is also
evidence that Simian virus 40 (SV40) may contribute to the
develop of mesothelioma, but the exact genetic alterations
leading to mesothelioma remain unknown (11, 13).
The aim of this study was to identify potential genes
involved in the pathogenesis of mesothelioma.
Materials and Methods
Cell lines. Mesothelioma cancer cell lines were obtained from the
following sources: LRK1A and REN through a generous gift from
Dr. Steven Albelda (University of Pennsylvania, Philadelphia, PA,
USA), NCI-H2052, H28, MSTO-211H, and H513 from American
Type Culture Collections (ATCC, Manassas, VA, USA), MS-1 and
NCI-H290 from NIH (Frederick, MD, USA) and LP9 were from
the Cell Culture Core Facility at Harvard University (Boston, MA,
USA). We should note that LP9 are mesothelial cells that are not
activated by SV40. All cell lines except LP9 were cultured in RPMI-
1640 supplemented with 10% fetal bovine serum, penicillin (100
IU/ml) and streptomycin (100 Ìg/ml). LP9 was cultured in M199
containing 15% medium plus 10 ng/ml EGF and 0.4 Ìg/ml HC. All
cells were cultured at 37ÆC in a humid incubator with 5% CO2.
Frozen cell pellets (–80ÆC) for use in the reference pool (WM-115,
NTERA-2, Colo 205, MCF7, Hs 578T, RPMI 8226, Hep G2, SW-872,
OVCAR-3, HL-60, & MOLT-4) were provided by the UCSF cancer
center microarray core facility in medium containing 10% DMSO.
Human tissues. Fresh mesothelioma tissues and adjacent normal
pleural tissues from patients undergoing primary resection of their
tumors were collected at the time of surgery and immediately snap-
frozen in liquid nitrogen (IRB H8714-22942-01). These tissue
samples were kept at –80ÆC in a liquid nitrogen freezer prior to use.
Final pathologic diagnosis was confirmed by a pathologist from the
University of California, San Francisco, USA. Patient identifiers
were coded to protect confidentiality.
cDNA microarray. The cDNA microarray chips used in this
experiment were from a print labeled Poly-L HPLower9K.7 and
were prepared by the UCSF Array Core Facility. These chips
Correspondence to: Jae Kim, MD, Jablons Lab, 2340 Sutter St., Rm
S341, University of California, San Francisco, San Francisco, CA
94115, U.S.A. Tel: +415 502 0555, Fax: +415 353 9530, e-mail:
Key Words: Stathmin, Oncoprotein 18, Op18, metablastin,
phosphoprotein 19, LAP18, mesothelioma.
ANTICANCER RESEARCH 27: 39-44 (2007)
Stathmin is Overexpressed in Malignant Mesothelioma
JAE Y. KIM1, CHANSONETTE HARVARD1, LIANG YOU1, ZHIDONG XU1,
KRISTOPHER KUCHENBECKER1, RICK BAEHNER2and DAVID JABLONS1
1Thoracic Oncology Laboratory, UCSF Comprehensive Cancer Center, San Francisco, CA 94115;
2Department of Pathology, UCSF, San Francisco, CA 94115, U.S.A.
contain approximately 8,600 ESTs. H28, MSTO-211H, H513, H290,
H2052, MS1, LRK1A, and LP9 cell lines were labeled with Cy5 and
run against a control reference pool labeled with Cy3. In subsequent
analysis, abnormal cell lines were compared to the LP9 cell line,
which was used as a control pleural cell line. Expression ratios were
generated by dividing ratios from averaged abnormal cell line runs
(n=3) by the ratios from the averaged LP9 runs (n=4).
Total RNA was extracted from the cell lines using an RNeasy
Mini or Midi kit (Qiagen, Valencia, CA, USA) according to the
manufacturer’s protocol. Cell pellets for the reference pool were
isolated using TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA).
10 Ìg of total RNA was used for first-strand cDNA synthesis for
Initially, 10 Ìg of total RNA was mixed with 5 Ìg oligo-(dT)18-20
and heated to 70ÆC for 10 min and then incubated on ice for 10 min.
To synthesize cDNA, a reverse transcription reaction was carried out
containing 10 Ìg of total RNA, 5 Ìg oligo (dT)18-20, 0.6 ÌL of 50x
aa-dUTP/dNTPs (30 mM 5-(3-aminoallyl)-2'-deoxyuridine-5'-
triophosphate aa-dUTP (Sigma), 50 mM each of dATP, dCTP,
dGTP, and 20 mM dTTP), 6 ÌL of 5X buffer, and 1.9 Ìl of 200 U/Ìl
SuperScript Reverse Transcriptase (Invitrogen). The reaction was
incubated for 2 h at 37ÆC.
Residual RNA was hydrolyzed with 0.5 mM EDTA and 1N
NaOH at 65ÆC for 15 min. The reaction was then neutralized by
the addition of 1M Tris-HCL. We used a Micron 30 concentrator
to remove residual Tris and concentrate the samples. The resulting
cDNA pellets were resuspended in 0.05 M NaHCO for 15 min at
room temperature and combined with NHS-ester Cy3 or Cy5
(Amersham, Piscataway, NJ, USA) monofunctional dye in DMSO.
Reactions were allowed to couple for 1 hour at room temperature
after which the reaction was quenched with the addition of 4M
hydroxylamine. Test samples labeled with Cy5 were combined with
the pooled reference labeled with Cy3 and the labeled products
purified using a QIAquick PCR purification system (Qiagen). The
probe mixture was heated at 100ÆC for 2 min, briefly centrifuged
and allowed to cool at room temperature for 5 min.
The probe was then injected under a cleaned lifterslip placed
gently over the cDNA array. Hybridizations were carried out in a
dual hybridization chamber (GeneMachines, San Carlos, CA,
USA) at 65ÆC for 6-8 h. Arrays were then washed in 2 x SSC/0.2%
SDS, followed by 0.1 x SSC. The microarrays were scanned with a
GenePix 4000B scanner and the images analyzed using GenePix
Pro 3.0 software (Axon Instruments, Union City, CA, USA).
RT-PCR. All previously noted cell lines were used for RT-PCR
assay. LP9 was used as a control pleural cell line. Total RNA was
extracted as previously noted. 0.5 Ìg of total RNA from each cell
line was used for RT-PCR reactions. Oncoprotein 18 primers were
obtained from Operon (Huntsville, AL, USA). GAPDH primers
were used to as a RNA quantity control. SuperScript One-Step RT-
PCR with Platinum Taq was used according to the manufacturer’s
protocol (Invitrogen). The PCR product was loaded onto 1%
agarose gels with ethidium bromide. After electrophoresis, the gels
were photographed under UV light.
Immunohistochemistry. Tumors were snap-frozen and embedded in
ornithine carbamyl transferase. Normal lung with adjacent pleural
tissues were used as control. They were cut into 4 to 5-mm sections,
dried at room temperature and fixed in acetone for 4ÆC for 10 min.
Tissues were incubated for 1 h at room temperature with stathmin
monoclonal antibody (Calbiochem, San Diego, CA, USA) at 1:200
dilution. After incubation with the primary antibody the tissue
sections were treated with 3% hydrogen peroxide and Normal Goat
Serum (Vector Labs, Burlingame, CA, USA). After the incubation
with the primary antibody, tissue sections were incubated with the
secondary biotinylated Goat anti-Rabbit (Vector Labs) followed by
avidin-biotin immuno-peroxidase. The sections were visualized using
diaminobenzidine chromogen (Sigma Aldrich, St. Louis, MO, USA)
and counterstained with hematoxylin (Thermo Shandon, Pittsburgh).
Western blot. Whole cells were homogenized and lysed with M-Per
mammalian protein extraction reagent for all cell lines and with
T-Per protein extraction reagent for all tissue samples (Pierce,
Rockford, IL, USA). Lysates were centrifuged for 14,000 xg for 5
min, and the supernatant was collected. The supernatant was
separated on 4-15% gradient SDS-polyacrylamide gels and
transferred to Immobilon-P (Millipore, Bedford, MA, USA)
membranes for Western blotting. Membranes were incubated with
stathmin antibody (Calbiochem, San Diego, CA, USA). Antigen-
antibody complexes were detected by enhanced chemiluminescence
blotting analysis system (Amersham Pharmacia Biotech,
Piscataway, NJ, USA). Beta-actin served as a loading control
(Sigma Chemical Co., St. Louis, MO, USA).
The cDNA expression array showed multiple genes were
overexpressed in mesothelioma cell lines compared to the
LP9 cell line (Table I). Stathmin was one of the most
strongly overexpressed genes. It was overexpressed in all 7
mesothelioma cell lines. The average level of expression was
5.4-fold higher than in the LP9 cell line (Figure 1).
Having detected stathmin overexpression by cDNA array,
we confirmed the results by RT-PCR. All mesothelioma cell
lines expressed stathmin more strongly than the LP9 cells
Having shown that stathmin mRNA levels were increased
in mesothelioma cell lines, we then showed that stathmin
protein levels were increased in the cell lines. Western blot
ANTICANCER RESEARCH 27: 39-44 (2007)
Table I. cDNA microarray analysis was performed on RNA extracted from
7 mesothelioma cell lines (LRK1A, H2052, 211H, H290, MS1, H513, and
H28) and compared to RNA from a normal pleural cell line (LP9). The
10 genes with the greatest ratios of expression (compared to LP9) are listed
with their mean ratio of expression (across all mesothelioma cell lines).
Gene Average ratio of expression
analysis revealed that stathmin was expressed in all malignant
mesothelioma cell lines, but not in LP9 cells (Figure 3). Next,
we showed that stathmin protein levels were increased in 7 of
8 mesothelioma tumor samples. Matched, normal pleural
tissue was unavailable for 4 of the tumor samples, but
stathmin protein was detected in all these tumor samples. In
contrast, none of the 4 matched, normal pleural tissues
expressed detectable levels of stathmin (Figure 4).
Finally, immunohistochemistry showed that mesothelioma
tissues stained for stathmin as well (Figure 5).
We have shown that stathmin is overexpressed in
mesothelioma. cDNA expression arrays showed that
stathmin mRNA levels are increased in all 7 mesothelioma
cell lines we tested. We also found stathmin protein levels to
be increased in all the cell lines and in 7 of 8 tumor tissues.
This was confirmed with immunohistochemistry. Although
we had a small sample number, these findings suggest that
stathmin overexpression is common in mesothelioma and
may play a role in its pathogenesis.
Stathmin, also known as oncoprotein 18, metablastin,
phosphoprotein 19 and LAP18, is a highly conserved
cytosolic phosphoprotein that helps regulate cell growth and
migration through regulation of microtubule stability (14).
Stathmin promotes microtubule depolymerization and
sequesters tubulin (15, 16). In addition to regulating cellular
progression through mitosis, stathmin plays a role in
mediating cell migration and perhaps metastasis (15).
Stathmin has been shown to be overexpressed in multiple
cancers, including leukemia, breast cancer, lung cancer,
hepatocellular cancer, ovarian cancer and prostate cancer (17-
23). To our knowledge, stathmin expression has not been
previously reported in mesothelioma and a PubMed search of
the keywords "stathmin and mesothelioma" revealed no hits.
Stathmin expression may be a marker for proliferation.
Curmi et al. showed that overexpression of stathmin correlates
with highly proliferative breast cancer tumors (18). Jeha et al.
showed that stathmin increases the proliferation rate of
leukemia cells (24). In hepatocellular cancer, stathmin
overexpression was found to be associated with larger tumor
size, tumor grade and higher stage. It also correlated with a
lower 5-year survival rate, independent of tumor stage (21).
Kim et al: Stathmin in Mesothelioma
Figure 1. cDNA microarray analysis was performed on RNA extracted from 7 mesothelioma cell lines and compared to RNA from a normal pleural cell
line (LP9). Stathmin was overexpressed in all 7 mesothelioma cell lines. LP9 expression level is defined as 1. All other expression levels are expressed as
ratios relative to LP9.
Stathmin overexpression may also confer chemotherapy
resistance. Two of the most commonly used classes of
chemotherapy drugs, vinka alkaloids and taxanes, interfere
with microtubule dynamics. Because stathmin also acts on
microtubules, there has been interest in its effect on resistance
to these drugs. Alli et al. found that stathmin overexpression in
breast epithelial cell lines decreased sensitivity to paclitaxel and
to vinblastine, and Rosell et al. found that stathmin expression
ANTICANCER RESEARCH 27: 39-44 (2007)
Figure 2. RT-PCR was performed on RNA isolated from 7 mesothelioma cell lines and a normal pleural cell line (LP 9). Stathmin was overexpressed
in all 7 mesothelioma cell lines. RT-PCR was performed using GAPDH primers as control.
Figure 3. Western blot analysis was performed on protein isolated from 8 mesothelioma cell lines and a normal pleural cell line (LP9). Proteins were
separated by SDS-PAGE and transferred to a membrane. They were then incubated with anti-stathmin antibody. Stathmin was overexpressed in all 8
mesothelial cell lines, but not in the normal pleura. Anti-beta-actin antibody was used as loading control.
correlated with response vinorelbine chemotherapy in non-
small cell lung cancer (25, 26). However, Nishio et al. found
that stathmin overexpression increases sensitivity to vindesine
(a vinka alkaloid) in lung cancer cell lines (27). Further studies
correlating stathmin expression with chemotherapy sensitivity
in mesothelioma may help guide therapy.
Our cDNA microarray identified multiple genes that were
overexpressed in mesothelioma cell lines. Due to the recent
studies implicating its role in proliferation and chemotherapy
resistance, we chose to focus on stathmin for the purposes
of this study. However, we are in the process of validating
the expression levels of other genes found to be either
overexpressed or underexpressed in mesothelioma tissues.
Due to the almost universally poor prognosis among
mesothelioma patients, correlation between stathmin
expression and clinical outcome could not be performed.
Kim et al: Stathmin in Mesothelioma
Figure 4. Western blot analysis was performed on protein isolated from 8 mesothelioma tumor samples and 4 matched, normal pleural samples (matched
normal pleura was unavailable for 4 of the tumor samples). Proteins were separated by SDS-PAGE and transferred to a membrane. They were then
incubated with anti-stathmin antibody. Stathmin was overexpressed in 7 of the 8 mesothelioma tumors, but not in any of the normal pleural tissue. Anti-
beta-actin antibody was used as loading control. T: tumor; N: matched non-tumor.
Figure 5. Immunohistochemistry. Mesothelioma tumor samples were snap frozen and cut into 4-5 Ìm sections and stained with anti-stathmin antibody.
The areas stained with stathmin are dark brown.
Our experiments showed increased levels of stathmin in
malignant mesothelioma, but did not address what role
stathmin plays in the actual pathogenesis of mesothelioma.
Future experiments could address this question by transfecting
LP9 cells with stathmin or by treating mesothelioma cells with
stathmin siRNA. Future studies on stathmin as a potential
therapeutic target in mesothelioma are warranted.
This work was partially supported by a National Institutes of
Health Grant (RO1 CA 093708-01A3), the Larry Hall and
Zygielbaum Memorial Trust, and the Kazan, McClain, Edises,
Abrams, Fernandez, Lyons & Farrise Foundations.
1 Pisick E and Salgia R: Molecular biology of malignant
mesothelioma: a review. Hematol Oncol Clin North Am 19:
Bang KM, Pinheiro GA, Wood JM and Syamlal G: Malignant
mesothelioma mortality in the United States, 1999-2001. Int J
Occup Environ Health 12: 9-15, 2006.
Hodgson JT, McElvenny DM, Darnton AJ, Price MJ and Peto
J: The expected burden of mesothelioma mortality in Great
Britain from 2002 to 2050. Br J Cancer 92: 587-593, 2005.
Peto J, Hodgson JT, Matthews FE and Jones JR: Continuing
increase in mesothelioma mortality in Britain. Lancet 345: 535-
Robinson BW and Lake RA: Advances in malignant
mesothelioma. N Engl J Med 353: 1591-1603, 2005.
Herndon JE, Green MR, Chahinian AP, Corson JM, Suzuki Y
and Vogelzang NJ: Factors predictive of survival among 337
patients with mesothelioma treated between 1984 and 1994 by
the Cancer and Leukemia Group B. Chest 113: 723-731, 1998.
Robledo R and Mossman B: Cellular and molecular
mechanisms of asbestos-induced fibrosis. J Cell Physiol 180:
Roggli VL, Sharma A, Butnor KJ, Sporn T and Vollmer RT:
Malignant mesothelioma and occupational exposure to
asbestos: a clinicopathological correlation of 1445 cases.
Ultrastruct Pathol 26: 55-65, 2002.
Papp T, Schipper H, Pemsel H, Bastrop R, Muller KM,
Wiethege T, Weiss DG, Dopp E, Schiffmann D and Rahman
Q: Mutational analysis of N-ras, p53, p16INK4a, p14ARF and
CDK4 genes in primary human malignant mesotheliomas. Int
J Oncol 18: 425-433, 2001.
10 Robinson BW, Musk AW and Lake RA: Malignant
mesothelioma. Lancet 366: 397-408, 2005.
11 Scagliotti GV and Novello S: State of the art in mesothelioma.
Ann Oncol 16 Suppl 2: ii240-245, 2005.
12 Lee AY, He B, You L, Dadfarmay S, Xu Z, Mazieres J,
Mikami I, McCormick F and Jablons DM: Expression of the
secreted frizzled-related protein gene family is down-regulated
in human mesothelioma. Oncogene 23: 6672-6676, 2004.
13 Gazdar AF and Carbone M: Molecular pathogenesis of
malignant mesothelioma and its relationship to simian virus 40.
Clin Lung Cancer 5: 177-181, 2003.
14 Rubin CI and Atweh GF: The role of stathmin in the
regulation of the cell cycle. J Cell Biochem 93: 242-250, 2004.
15 Iancu-Rubin C and Atweh GF: p27(Kip1) and stathmin share
the stage for the first time. Trends Cell Biol 15: 346-348, 2005.
16 Baldassarre G, Belletti B, Nicoloso MS, Schiappacassi M,
Vecchione A, Spessotto P, Morrione A, Canzonieri V and
Colombatti A: p27(Kip1)-stathmin interaction influences
sarcoma cell migration and invasion. Cancer Cell 7: 51-63, 2005.
17 Brattsand G: Correlation of oncoprotein 18/stathmin
expression in human breast cancer with established prognostic
factors. Br J Cancer 83: 311-318, 2000.
18 Curmi PA, Nogues C, Lachkar S, Carelle N, Gonthier MP,
Sobel A, Lidereau R and Bieche I: Overexpression of stathmin
in breast carcinomas points out to highly proliferative tumours.
Br J Cancer 82: 142-150, 2000.
19 Melhem R, Hailat N, Kuick R and Hanash SM: Quantitative
analysis of Op18 phosphorylation in childhood acute leukemia.
Leukemia 11: 1690-1695, 1997.
20 Chen G, Wang H, Gharib TG, Huang CC, Thomas DG,
Shedden KA, Kuick R, Taylor JM, Kardia SL, Misek DE,
Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM and
Beer DG: Overexpression of oncoprotein 18 correlates with
poor differentiation in lung adenocarcinomas. Mol Cell
Proteomics 2: 107-116, 2003.
21 Yuan RH, Jeng YM, Chen HL, Lai PL, Pan HW, Hsieh FJ,
Lin CY, Lee PH and Hsu HC: Stathmin overexpression
cooperates with p53 mutation and osteopontin overexpression,
and is associated with tumour progression, early recurrence,
and poor prognosis in hepatocellular carcinoma. J Pathol, in
22 Price DK, Ball JR, Bahrani-Mostafavi Z, Vachris JC, Kaufman
JS, Naumann RW, Higgins RV and Hall JB: The
phosphoprotein Op18/stathmin is differentially expressed in
ovarian cancer. Cancer Invest 18: 722-730, 2000.
23 Friedrich B, Gronberg H, Landstrom M, Gullberg M and
Bergh A: Differentiation-stage specific expression of
oncoprotein 18 in human and rat prostatic adenocarcinoma.
Prostate 27: 102-109, 1995.
24 Jeha S, Luo XN, Beran M, Kantarjian H and Atweh GF:
Antisense RNA inhibition of phosphoprotein p18 expression
abrogates the transformed phenotype of leukemic cells. Cancer
Res 56: 1445-1450, 1996.
25 Alli E, Bash-Babula J, Yang JM and Hait WN: Effect of
stathmin on the sensitivity to antimicrotubule drugs in human
breast cancer. Cancer Res 62: 6864-6869, 2002.
26 Rosell R, Scagliotti G, Danenberg KD, Lord RV, Bepler G,
Novello S, Cooc J, Crino L, Sanchez JJ, Taron M, Boni C, De
Marinis F, Tonato M, Marangolo M, Gozzelino F, Di
Costanzo F, Rinaldi M, Salonga D and Stephens C:
Transcripts in pretreatment biopsies from a three-arm
randomized trial in metastatic non-small-cell lung cancer.
Oncogene 22: 3548-3553, 2003.
27 Nishio K, Nakamura T, Koh Y, Kanzawa F, Tamura T and
Saijo N: Oncoprotein 18 overexpression increases the
sensitivity to vindesine in the human lung carcinoma cells.
Cancer 91: 1494-1499, 2001.
Received November 13, 2006
Accepted November 17, 2006
ANTICANCER RESEARCH 27: 39-44 (2007)