Sprouty2 protein enhances the response to gefitinib through epidermal growth factor receptor in colon cancer cells.
ABSTRACT Sprouty2 (Spry2) is known to increase the expression of epidermal growth factor receptors (EGFR) by conjugating with c-Casitas B-lineage lymphoma (C-Cbl) to decrease protein degradation. The effect of Spry2 on the treatment of gefitinib, a tyrosine kinase inhibitor of EGFR, with regards to colon cancer is still unclear. The half maximal inhibitory concentration (IC50) values of gefitinib in six colon cancer cell lines were assessed. HCT116 and C2BBel cells expressed lower levels of Spry2 protein and were less sensitive to gefitinib, whereas HT29 cells that expressed high levels of Spry2 protein were more sensitive to gefitinib. The sensitivity to gefitinib was increased after overexpression of Spry2 in HCT116 cells, whereas it was decreased after Spry2 knockdown in HT29 cells. The levels of both phosphorylated and total EGFR were increased when HCT116 cells ectopically overexpressed Spry2, with concomitant increase in phosphatase and tensin homolog (PTEN) expression. Inhibition of EGFR by cetuximab reduced sensitivity to gefitinib in HCT116 cells overexpressing Spry2. However, knockdown of PTEN or K-ras failed to diminish the effect of Spry2 on gefitinib sensitivity. Of note, Spry2 enhanced the antitumor effect of gefitinib in a xenograft model of HCT116 tumors, which harbored K-ras codon 13 mutation. In conclusion, Spry2 can enhance the response of colon cancer cells to gefitinib by increasing the expression of phosphorylated and total EGFR. These results suggest that Spry2 may be a potential biomarker in predicting the response to anti-EGFR treatment in colon cancer and that it is necessary to conduct clinical studies to incorporate Spry2 into the network of cancer treatment.
- [show abstract] [hide abstract]
ABSTRACT: Sprouty family proteins are novel regulators of growth factor actions. Human Sprouty 2 (hSPRY2) inhibits the proliferation of a number of different cell types. However, the mechanisms involved in the anti-proliferative actions of hSPRY2 remain to be elucidated. Here we have demonstrated that hSPRY2 increases the amount of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and decreases its phosphorylation. The resultant increase in PTEN activity is reflected in decreased activation of Akt by epidermal growth factor and serum. Consistent with increased PTEN activity, in hSPRY2-expressing cells, the progression of cells from the G1 to S phase is decreased. By using PTEN null primary mouse embryonic fibroblasts and their isogenic controls as well as small interfering RNA against PTEN, we demonstrated that PTEN is necessary for hSPRY2 to inhibit Akt activation by epidermal growth factor as well as cell proliferation. Overall, we concluded that hSPRY2 mediates its anti-proliferative actions by altering PTEN content and activity.Journal of Biological Chemistry 03/2006; 281(8):4816-22. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The anti-epidermal growth factor receptor (anti-EGFR) cetuximab has been proven to be efficient in metastatic colorectal cancer. The molecular mechanisms underlying the clinical response to this drug remain unknown. Genetic alterations of the intracellular effectors involved in EGFR-related signaling pathways may have an effect on response to this targeted therapy. In this study, tumors from 30 metastatic colorectal cancer patients treated by cetuximab were screened for KRAS, BRAF, and PIK3CA mutation by direct sequencing and for EGFR copy number by chromogenic in situ hybridization. Eleven of the 30 patients (37%) responded to cetuximab. A KRAS mutation was found in 13 tumors (43%) and was significantly associated with the absence of response to cetuximab (KRAS mutation in 0% of the 11 responder patients versus 68.4% of the 19 nonresponder patients; P = 0.0003). The overall survival of patients without KRAS mutation in their tumor was significantly higher compared with those patients with a mutated tumor (P = 0.016; median, 16.3 versus 6.9 months). An increased EGFR copy number was found in 3 patients (10%) and was significantly associated with an objective tumor response to cetuximab (P = 0.04). In conclusion, in this study, KRAS mutations are a predictor of resistance to cetuximab therapy and are associated with a worse prognosis. The EGFR amplification, which is not as frequent as initially reported, is also associated with response to this treatment.Cancer Research 05/2006; 66(8):3992-5. · 8.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Sprouty (Spry) proteins were found to be endogenous inhibitors of the Ras/mitogen-activated protein kinase pathway that play an important role in the remodeling of branching tissues. We investigated Spry expression levels in various cancers and found that Spry1 and Spry2 were down-regulated consistently in breast cancers. Such prevalent patterns of down-regulation may herald the later application of these isoforms as tumor markers that are breast cancer specific and more profound than currently characterized markers. Spry1 and 2 were expressed specifically in the luminal epithelial cells of breast ducts, with higher expression during stages of tissue remodeling when the epithelial ducts are forming and branching. These findings suggest that Sprys might be involved as a modeling counterbalance and surveillance against inappropriate epithelial expansion. The abrogation of endogenous Spry activity in MCF-7 cells by the overexpression of a previously characterized dominant-negative mutant of Spry, hSpry2Y55F resulted in enhanced cell proliferation in vitro. The hSpry2Y55F stably expressing cells also formed larger and greater number of colonies in the soft-agar assay. An in vivo nude mice assay showed a dramatic increase in the tumorigenic potential of hSpry2Y55F stable cells. The consistent down-regulation of Spry1 and 2 in breast cancer and the experimental evidence using a dominant-negative hSpry2Y55F indicate that Spry proteins may actively maintain tissue integrity that runs amok when their expression is decreased below normal threshold levels. This alludes to a previously unrecognized role for Sprys in cancer development.Cancer Research 10/2004; 64(17):6127-36. · 8.65 Impact Factor
Sprouty2 protein enhances the response to gefitinib
through epidermal growth factor receptor in colon
Yin-Hsun Feng,1,2Chao-Jung Tsao,3Chao-Liang Wu,1,4Jan-Gowth Chang,5Pei-Jung Lu,1Kun-Tu Yeh,6
Gia-Shing Shieh,1Ai-Li Shiau1,7,9and Jeng-Chang Lee8,9
1Graduate Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan;2Department of Hematology and
Oncology, Chi-Mei Medical Center, Yong Kang City, Tainan, Taiwan;3Department of Hematology and Oncology, Chi-Mei Medical Center, Liouying
Township, Tainan, Taiwan;4Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan;
5Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;6Department of Surgical Pathology, Changhua Christian
Hospital, Changhua, Taiwan;7Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan;
8Department of Surgery, College of Medicine, National Cheng Kung University, Tainan, Taiwan
(Received February 16, 2010 ⁄Revised May 27, 2010 ⁄Accepted May 28, 2010 ⁄ Accepted manuscript online June 7, 2010 ⁄Article first published online July 8,
Sprouty2 (Spry2) is known to increase the expression of epidermal
growth factor receptors (EGFR) by conjugating with c-Casitas
B-lineage lymphoma (C-Cbl) to decrease protein degradation.
The effect of Spry2 on the treatment of gefitinib, a tyrosine kinase
inhibitor of EGFR, with regards to colon cancer is still unclear. The
half maximal inhibitory concentration (IC50) values of gefitinib in
six colon cancer cell lines were assessed. HCT116 and C2BBel cells
expressed lower levels of Spry2 protein and were less sensitive to
gefitinib, whereas HT29 cells that expressed high levels of Spry2
protein were more sensitive to gefitinib. The sensitivity to
gefitinib was increased after overexpression of Spry2 in HCT116
cells, whereas it was decreased after Spry2 knockdown in HT29
cells. The levels of both phosphorylated and total EGFR were
increased when HCT116 cells ectopically overexpressed Spry2, with
concomitant increase in phosphatase and tensin homolog (PTEN)
expression. Inhibition of EGFR by cetuximab reduced sensitivity to
gefitinib in HCT116 cells overexpressing Spry2. However, knock-
down of PTEN or K-ras failed to diminish the effect of Spry2 on
gefitinib sensitivity. Of note, Spry2 enhanced the antitumor
effect of gefitinib in a xenograft model of HCT116 tumors, which
harbored K-ras codon 13 mutation. In conclusion, Spry2 can
enhance the response of colon cancer cells to gefitinib by increas-
ing the expression of phosphorylated and total EGFR. These results
suggest that Spry2 may be a potential biomarker in predicting the
response to anti-EGFR treatment in colon cancer and that it is
necessary to conduct clinical studies to incorporate Spry2 into the
network of cancer treatment. (Cancer Sci 2010; 101: 2033–2038)
147 000 newly diagnosed cases of colorectal cancer and nearly
50 000 deaths associated with this disease.(1)Up to 20% of new
cases present with metastatic disease. Among the patients who
present with localized disease, approximately 20% will subse-
quently relapse with distant metastasis. Over little more than a
decade, the options for systemic therapy have progressed signifi-
cantly from 5-fluorouracil (5-FU) alone in 1995 to an armamen-
tarium of several chemotherapy and biological agents.(2)Among
these biological agents, epidermal growth factor receptor
(EGFR)-targeted therapy is one of the important clinical strate-
gies to apply in patients with metastatic colon cancer.
Epidermal growth factor receptor (EGFR) is a 170-kDa trans-
membrane glycoprotein, with an extracellular ligand-binding
domain and an intracellular region containing the tyrosine
kinase domain.(3)Epidermal growth factor receptor (EGFR)
activation on the cancer cell surface is believed to promote cell
olorectal cancer is the second leading cause of death from
cancer in the USA. It is estimated that there will be
growth, differentiation, cell survival, drug and radiation sensitiv-
ity, and angiogenesis. Expression of a high level of EGFR has
been associated with a poorer prognosis in colon cancer patients
undergoing curative surgery.(4)Novel anticancer drugs targeting
the EGFR family have been applied in treating various types of
human cancers, including colon cancer, lung cancer, breast can-
cer, and squamous cell carcinoma of head and neck. These strat-
egies targeting EGFR include monoclonal antibodies that block
the extracellular ligand binding domain of the receptor such as
cetuximab and low molecular weight tyrosine kinase inhibitors
(TKI), such as gefitinib and erlotinib.
Gefitinib (Iressa, ZD-1839) is a synthetic anilinoquinazoline
and orally active selective EGFR-TKI that blocks the signal
transduction pathway implicated in the proliferation and survival
of cancer cells. Disappointingly, the emerging clinical experi-
ence across a range of cancer types reveals that despite the anti-
EGFR agents demonstrating some antitumor activity, there is a
high level of de novo resistance to such treatment.(5–7)Several
biomarkers have been demonstrated to exert a predictive value
in anti-EGFR treatment. Specific mutations in the EGFR gene
are correlated with clinical response to gefitinib in patients with
non-small-cell lung cancer.(8)Phospho-EGFR level and high
serum EGFR at baseline increase the sensitivity of colon cancer
cells to gefitinib.(9,10)There is emerging evidence that K-ras
mutation serves as a major predictor of resistance to cetuximab
in colon cancer.(11,12)Furthermore, loss of phosphatase and ten-
sin homolog (PTEN) protein has been shown to be associated
with nonresponsiveness to cetuximab.(13)
The Sprouty (Spry) protein was first described by Hacohen
et al.(14)as an inhibitor of fibroblast growth factor-stimulated
tracheal branching during Drosophila development. Four mam-
malian Spry genes have been defined based on sequence similar-
ity with Drosophila Spry. Sprouty2 (Spry2) encodes 32- to
34-kDa proteins that share a highly conserved carboxyl-terminal
cysteine-rich spry domain. Spry2 is known to be involved in
EGFR pathway. C-Cbl is a prominent binding partner of Spry2.
Cbl proteins function as ubiquitin ligases which through mono-
and poly-ubiquitination of receptor tyrosine kinases (RTKs) ini-
tiate their endocytosis and proteosomal degradation. Spry2 has
been shown to abrogate EGFR ubiquitylation and endocytosis,
and sustain epidermal growth factor (EGF)-induced extra-
cellular signal-regulated kinase (ERK) signaling.(15)Later, it was
reported that Spry2 induces down-regulation of EGFR at the
9To whom correspondence should be addressed.
E-mail: firstname.lastname@example.org; email@example.com
ª ª 2010 Japanese Cancer Association
| vol. 101| no. 9|
Cbl⁄CIN85 interface and interferes with the trafficking of
activated EGFR specifically at the step of progression from
early to late endosomes.(16,17)However, it is still unclear whether
expression of Spry2 affects the efficacy of gefitinib treatment in
colon cancer. In this study, we demonstrate that expression of
Spry2 increases sensitivity to gefitinib therapy in colon cancer.
Materials and Methods
Reagents. Gefitinib was provided by AstraZeneca (Wilming-
ton, DE, USA). A 10 mmol⁄L working solution in DMSO was
prepared and stored at )20?C. Cetuximab was obtained from
Merck (Darmstadt, Germany).
Cell culture. C2BBel, LS174T, HCT116, and HT29 human
colon cancer cell lines were purchased from the Food Industry
Research and Development Institute (Hsinchu, Taiwan). SW620
and SW480 human colon cancer cell lines were obtained from
the American Type Culture Collection (Rockville, MD, USA).
Cell viability assay. Cells were seeded at 10 000 per well in
96-well plates. They were treated with increasing doses of gefiti-
nib for 72 h. Cell viability was assessed by the MTT assay
(Sigma Aldrich, St. Louis, MO, USA). The absorbance was
measured at 570 nm on the MRX Revelation microtiter plate
reader (Dynex Technologies, Chantilly, VA, USA). The half
maximal inhibitory concentration (IC50) was calculated with
SigmaPlot 9.0 statistical software (Jandel Scientific, Corte
Madera, CA, USA). Data were expressed as the ratio of cell
numbers relative to the controls. Each value represents the
mean ± SD of at least three determinations.
Plasmids, transfection, and stable clones. The pSpry2-EGFP-
N3 plasmid encoding Spry2-EGFP fusion protein was obtained
from G.F. Vande Woude (University of California, Irvine, CA,
USA).(18)HCT116 cells were transfected with pSpry2-EGFP-
N3 or empty vector pEGFP-N3 (Clontech, Palo Alto, CA, USA)
with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
G418 was used for selection of a stable clone overexpressing
Spry2 (HCT116⁄Spry2) and a control clone (HCT116⁄v).
Lentivirus infection and shRNA knockdown. A set of five
pLKO.1-puro-based lentiviral vectors containing stem-loop cas-
settes encoding shRNAs for human Spry2 (TRCN 0000007520-
7524) (designated Spry2 shRNA #1–5), and similar vectors
encoding shRNAs for PTEN (TRCN 0000002747), K-ras (TRCN
0000033260), and luciferase (Luc) (TRCN 0000072246) were
Taiwan. Knockdown efficiencies were assessed, and two Spry2
shRNA (#1 and #2) were selected for further production of
recombinant lentiviruses. Recombinant lentiviruses were pro-
duced by transient transfection of 293T cells with pLKO.1-puro
constructs along with the packaging construct psPAX2 and the
VSV-G expression construct pMD2G using the calcium phos-
Immunoblotting. Total cell lysates were harvested for immu-
noblot analysis using anti-Spry2 (Upstate Biotechnology, Lake
Placid, NY, USA), anti-b-actin (Sigma Aldrich), anti-phospho-
EGFR, anti-EGFR, and anti-PTEN (Cell Signaling Technolo-
gies, Beverly, MA, USA) antibodies as primary antibodies.
Horseradish peroxidase-conjugated antirabbit IgG (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) and antimouse IgG
(Santa Cruz Biotechnology) were used as secondary antibodies
where appropriate, and protein-antibody complexes were visual-
ized using the ECL system (Amersham Biosciences, Piscataway,
Reverse transcription–polymerase chain reaction (RT-PCR). Total
RNA was extracted from colon cancer cells using RNA
extraction kits (Qiagen, Hilden, Germany). RNA was treated
with RNase-free DNase for 30 min at room temperature. First-
strand cDNA was synthesized using 2 lg of DNase-treated
RNA, 1 lL of oligo dT, and SuperScript III RT with the Super-
Script First-Strand Synthesis System (Invitrogen). Polymerase
chain reaction (PCR) was performed with 0.3 lg of cDNA.
The primers used for PCR were H-PTEN primer 1 (5¢-
CTCCAATTCAGGACCCACACGAC-3¢), H-PTEN primer 2
(5¢-AAGTACAGCTTCACCTTAAA-3¢), and H-PTEN primer 3
(5¢-CGGGAAGACAAGTTCATGTAC-3¢). The amplification
condition was used as described previously.(20)For determina-
tion of K-ras mRNA expression, the primers used for K-ras were
H-K-ras primer 1 (5¢-GGGTGTTGATGCCTTCC-3¢) and H-K-
ras primer 2 (5¢-AATCAACTGCATGCACCAAA-3¢). The
amplification condition was 32 cycles of 1 min at 94?C for
denaturation, 30 s at 55?C for annealing, and 1 min at 72?C for
extension. The products were subjected to electrophoresis on a
2% agarose gel, and the DNA was visualized by ethidium bro-
Animal study. Animal care in accordance with institutional
guidelines and authority to perform in vivo work was granted by
the Home Office (project license 97022902, Chi-Mei Medical
Center). HCT116⁄Spry2 and HCT116⁄v xenografts were estab-
lished by injection of cancer cells into 7- to 8-week-old BALB-
C athymic female nude mice purchased from the National Labo-
ratory Animal Center, Taiwan. Groups of five nude mice were
inoculated subcutaneously into both flanks with HCT116⁄Spry2
or HCT116⁄v cells (2 · 106⁄injection site). Gefitinib dissolved
in carrier solution (0.1% aqueous TWEEN 80; Sigma-Aldrich)
was administered to all mice at a dose of 50 mg⁄kg by oral
gavage on days 1–5 and 8–12.(21)Tumors were measured every
5 days and calculated as p⁄6 · (tumor width)2· tumor length.
Mice with tumors exceeding 17 mm diameter were culled in
accordance with the guideline.
Immunohistochemistry. Cryostat sections (10 lm) fixed in
4% paraformaldehyde were incubated for 1 h with hSpry2 rabbit
polyclonal antibodies (Upstate Biotechnology). After wash, the
slides were incubated for 30 min with the DAKO EnVision Sys-
tem (Dako, Carpinteria, CA, USA). Antibody staining was done
by treatment with diaminobenzidine. Sections were counter-
stained with Mayer’s hematoxylin. All of the incubation and
staining steps were done at room temperature. Negative control
slides processed without primary antibody were included for
Statistics. Statistical significance
assessed with the Student’s t-test. Any P-value of <0.05 was
regarded statistically significant.
Expression of Spry2 protein positively correlates with the
sensitivity of colon cancer cells to gefitinib. To test the influence
of Spry2 expression on the effect of gefitinib, we assessed the
expression of Spry2 protein in six colon cancer cell lines.
Among these cancer cell lines, C2BBel and HCT116 cells
expressed lower levels of Spry2 protein (Fig. 1b) and were
highly resistant togefitinib,
14.72 ± 3.18 lM for C2BBel and 9.52 ± 0.59 lM for HCT116
(Fig. 1a). In contrast, HT29 cells that expressed high levels of
Spry2 protein (Fig. 1b) were sensitive to gefitinib, with an IC50
value of 2.78 ± 0.11 lM (Fig. 1a). The remaining cell lines,
including SW620, SW480, and LS174T, which expressed mod-
erate levels of Spry2 protein (Fig. 1b), exhibited moderate resis-
tance to gefitinib (Fig. 1a,c).
Overexpression of Spry2 enhances the sensitivity of colon
cancer cells to gefitinib. We next used HCT116⁄Spry2 cells sta-
bly overexpressing Spry2 and HCT116⁄v control cells to verify
the correlation between Spry2 expression and gefitinib sensitiv-
ity. Overexpression of Spry2 enhanced the sensitivity of
HCT116 cells to gefitinib compared with the HCT116⁄v cells
(Fig. 2a). The IC50 values of gefitinib were 8.34 ± 2.60 lM in
HCT116⁄v cells and 3.22 ± 0.23 lM in HCT116⁄Spry2 cells.
with IC50values of
ª ª 2010 Japanese Cancer Association
Reciprocally, shRNA knockdown of Spry2 expression rendered
HT29 cells more resistant to gefitinib (Fig. 2b).
Overexpression of Spry2 enhances the expression of phos-
phorylated EGFR, total EGFR, and PTEN in colon cancer cells. It
has been reported that phosphorylated EGFR and PTEN regulate
the sensitivity of colon cancer cells to anti-EGFR treatment.
HCT116 cells were used to assess the effect of Spry2 on the
expression of EGFR and its downstream effectors. Both phos-
phorylated and total EGFR as well as PTEN levels were
increased in HCT116 cells overexpressing Spry2 in the absence
of gefitinib (Fig. 3a). In the presence of gefitinib, overexpression
of Spry2 also activated EGFR and up-regulated PTEN expres-
sion in HCT116 cells (Fig. 3b). Collectively, these results sug-
gest that increased sensitivity of colon cancer cells to gefitinib
by Spry2 overexpression might be dependent on the EGFR
status and PTEN expression.
Inhibition of EGFR by cetuximab counteracts Spry2-induced
gefitinib sensitivity in colon cancer cells. To confirm whether
EGFR expression induced by Spry2 mediates gefitinib sensitiv-
ity, HCT116⁄Spry2 cells were treated with 20 lg⁄mL of cetux-
imab for 24 h as previously described.(22)Figure 4(a) shows that
pretreatment with cetuximab rendered cells more resistant to
gefitinib. Several downstream effectors of EGFR including
K-ras and PTEN have been explored to predict for response to
gefitinib. To clarify whether PTEN expression is associated with
sensitivity to gefitinib in HCT116⁄Spry2 cells, we knocked
down PTEN expression by lentivirus-mediated delivery of
PTEN shRNA. Figure 4(b) shows that the IC50 values of gefiti-
nib were similar regardless of PTEN silencing, implying that the
increased sensitivity of colon cancer cells to gefitinib induced
by Spry2 is independent of PTEN expression. Next, we charac-
terized the K-ras mutations in the six colon cancer cell lines.
Except for HT29 and C2BBel cells, four colon cancer cells har-
bored K-ras mutations, as confirmed by a K-ras mutation detec-
tion assay (Fig. S1a). DNA auto-sequencing confirmed that
LS174T, SW620, and SW480 cells had codon 12, and HCT116
cells had codon 13 K-ras mutations (Fig. S1b). The impact of
K-ras mutation on gefitinib sensitivity enhanced by Spry2 was
assessed in SW620 cells using shRNA knockdown. Figure 4(c)
reveals that knockdown of K-ras in SW620 cells did not affect
their sensitivity to gefitinib.
Sprouty2 (Spry2) enhances the antitumor activity of gefitinib
in the HCT116 xenograft model. To confirm the effect of Spry2
on gefitinib treatment in colon cancer, HCT116 cells were
implanted in nude mice with or without ectopic expression of
Spry2 accompanied with oral administration of gefitinib. As
treated with increasing doses of gefitinib for 72 h, and cell viability was determined by the MTT assay. Each value represents the mean of three
determinations. Bar, SD. (b) Expression levels of Spry2 protein in various colon cancer cells detected by immunoblot analysis. Values shown
below the blot are the ratios between the intensity of the bands corresponding to Spry2 and b-actin determined by densitometry. (c) The half
maximal inhibitory concentration (IC50) values of gefitinib in different colon cancer cell lines calculated from (a).
Sprouty2 (Spry2) expression in colon cancer cell lines correlates with their sensitivity to gefitinib. (a) Various colon cancer cells were
stably overexpressing Spry2. The IC50 values of gefitinib were 3.22 ± 0.23 lM in HCT116⁄Spry2 cells and 8.34 ± 2.60 lM in control HCT116⁄v
cells. (b) Cytotoxic effect of gefitinib on HT29 cells after Spry2 knockdown by two Spry2 shRNA lentiviruses. Two Spry2 knockdown cells were
less sensitive to gefitinib compared with Luc knockdown cells serving as the control.
Sprouty2 (Spry2) expression affects the sensitivity of colon cancer cells to gefitinib. (a) Cytotoxic effect of gefitinib on HCT116⁄Spry2 cells
Feng et al. Cancer Sci|
| vol. 101| no. 9|
ª ª 2010 Japanese Cancer Association
shown in Figure 5(a), seven of 10 implants of HCT116⁄v cells
generated tumors despite gefitinib treatment. By marked con-
trast, only two of 10 implants of HCT116⁄Spry2 cells formed
tumors. Immunohistochemical analysis also confirms that
tumors derived from HCT116⁄Spry2 cells expressed higher lev-
els of Spry2 than did tumors derived from HCT116⁄v cells
(Fig. 5b). Notably, HCT116⁄v cells formed larger tumors than
HCT116⁄Spry2 cells, indicating that Spry2 expression may con-
tribute to the enhanced antitumor effect of gefitinib against
colon cancer in animal models.
Gefitinib and erlotinib are EGFR-TKIs commonly used in Asian
patients with lung cancer. Mutations of EGFR tyrosine kinase
have been shown to predict better response to TKIs as well as to
improve the survival of lung cancer patients.(23,24)Phase I⁄II tri-
als in patients with metastatic colon cancer showed little activ-
ity; but preclinical studies suggested a supra-additional growth
inhibitory effect of gefitinib when combined with different cyto-
toxic drugs, which provided the rationale for several clinical tri-
als of gefitinib in combination with chemotherapy in patients
with metastatic colon cancer.(25,26)The important mutations
associated with the activity of gefitinib are EGFR tyrosine
kinase mutations in exons 18, 19, and 21(8,27); however, this
seems not to be the case in metastatic colon cancer, as these
mutations are not so commonly found in colon cancer. It was
reported that none of the 11 colon cancer cell lines examined
have mutated EGFR, and only 12% of colon cancer patients
have somatic mutations of EGFR according to a Japanese
study.(28)It is worth exploring the molecular network to predict
the response to gefitinib in further colon cancer therapy.
Spry2 is a well-known tumor suppressor in many human can-
cers, such as breast, lung, prostate, and liver cancers.(28–32)Our
results also demonstrated that overexpression of Spry2 reduces
cell proliferation, colony formation potential, and migration of
colon cancer cells, as well as suppresses cancer growth in vivo,
which reflects the role of Spry2 as a tumor suppressor in colon
cancer (Feng et al., unpublished manuscript, 2010). Spry2
can enhance the expression of EGFR through decreasing EGFR
degradation in cancer cells. In HeLa cells, overexpression of
Spry2 increases the total amount of PTEN but decreases the
amount of phosphorylated PTEN.(33)The resultant increase in
PTEN activity leads to decreased activation of AKT and down-
stream signaling. Interestingly, Spry2 is tightly connected to the
EGFR pathway which inspired our investigation to define its
role in gefitinib therapy. Our data show that the expression of
Spry2 is associated with a better therapeutic effect of gefitinib in
colon cancer cells. These results indicate that Spry2 could be a
potential target to predict or manipulate gefitinib response for
colon cancer. Because K-ras mutations are also important in pre-
dicting response to anti-EGFR treatment in colon cancer, it is
mandatory to clarify the status of K-ras mutations in colon can-
cer cells. In the present study, all of the colon cancer cell lines
tested, except HT29 and C2BBel cells, had K-ras mutations.
HT29 cells which do not harbor K-ras mutation became more
resistant to gefitinib after their endogenous Spry2 was sup-
pressed by shRNA knockdown (Fig. 2b). However, silencing of
K-ras in SW620 cells did affect their sensitivity to gefitinib
(a) (b) (c)
enhanced by Sprouty2 (Spry2). (a) HCT116⁄Spry2 cells that had been treated with or without 20 lg⁄mL of cetuximab for 24 h were exposed to
different doses of gefitinib for 72 h. Cell viability was determined by the MTT assay. The IC50 values of gefitinib were 9.17 ± 2.00 lM in
cetuximab-treated cells and 4.31 ± 0.49 lM in cells without cetuximab treatment. (b) HCT116 ⁄Spry2 cells were infected with lentiviral vectors
encoding phosphatase and tensin homolog (PTEN) shRNA or Luc shRNA serving as the control for 24 h. Cells were then treated with different
doses of gefitinib for 72 h. Cell viability was determined by the MTT assay. The IC50 values of gefitinib were 3.84 ± 0.68 lM for PTEN
knockdown cells and 3.23 ± 0.24 lM for Luc knockdown cells. (c) SW620 cells were infected with lentiviral vectors encoding K-ras shRNA or Luc
shRNA serving as the control for 24 h. Cells were then treated with different doses of gefitinib for 72 h. Cell viability was determined by the
MTT assay. The IC50 values of gefitinib were 3.86 ± 0.71 lM for K-ras knockdown cells and 3.56 ± 0.76 lM for Luc knockdown cells. The
efficiency of PTEN (b) and K-ras (c) knockdown was assessed by RT-PCR analysis for the expression of PTEN (b) and K-ras (c) as well as GAPDH
serving as the loading control (b,c).
Inhibition of epidermal growth factor receptor (EGFR) by cetuximab reduces the sensitivity of HCT116 colon cancer cells to gefitinib
phosphorylated and total epidermal growth factor receptor (EGFR) as
well as phosphatase and tensin homolog (PTEN) in HCT116 colon
cancer cells. (a) HCT116 cells were transfected with 5 lg of pSpry2-
GFP-N3 plasmid, and total cell lysates were immunoblotted 48 h later
for expression of phosphorylated EGFR (p-EGFR), total EGFR, PTEN,
Spry2, and b-actin. Detection of Spry2-GFP and Spry2 proteins
corresponds to the ectopic and endogenous expression of Spry2,
respectively. (b) HCT116 cells were pretreated with 10 lM of gefitinib
for 24 h after transfection with pSpry2-GFP-N3 plasmid. Total cell
lysates were analyzed by immunoblotting after 48 h for expression of
indicated proteins as described in (a).
Overexpression of Sprouty2 (Spry2) enhances the levels of
ª ª 2010 Japanese Cancer Association
(Fig. 4c). The findings of an insignificant role of K-ras in gefiti-
nib treatment of colon cancer cells are consistent with the results
from clinical trials in lung cancer.(34,35)This implies that Spry2
affects gefitinib sensitivity in a K-ras mutation-independent
manner. Reduction of PTEN protein is associated with natural
resistance to gefitinib,(36)and restoration of PTEN induces sig-
nificant growth inhibition in gefitinib-resistant lung cancer
cells.(37)Phosphatase and tensin homolog (PTEN) expression
was enhanced after HCT116 cells were forced to express Spry2
in our study. However, we failed to show that knockdown of
PTEN could reduce the effect of Spry2 on gefitinib sensitivity in
colon cancer cells. Further investigations are warranted to define
the association between PTEN and Spry2 in gefitinib treatment.
Our failure to identify any association between K-ras mutation
or PTEN expression and gefitinib sensitivity in colon cancer
using the RNA interference approach does not exclude the pos-
sibility that such a correlation may exist.
In summary, we have found that the overexpression of
Spry2 enhances the sensitivity of colon cancer cells to gefitinib
in vitro. For further validation of this correlation, we have also
shown that overexpression of Spry2 in HCT116 cells leads to
dramatic decreases in both tumor incidence and size in com-
parison to the control HCT116 cells in nude mice. These results
suggest that Spry2 may play a role in gefitinib sensitivity in
addition to EGFR mutations. However, more clinical studies
involving the correlation of expression of Spry2 and EGFR as
well as gefitinib sensitivity in colon cancer are necessary to
guide the clinical implications.
This work was supported by grants from National Cheng Kung Univer-
sity Hospital and Chi-Mei Medical Center, Liouying, Taiwan (CLFHR
9724 and CLFHR 9833).
The authors have no conflict of interest.
1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009.
CA Cancer J Clin 2009; 59: 225–49.
2 Segal NH, Saltz LB. Evolving treatment of advanced colon cancer. Annu Rev
Med 2009; 60: 207–19.
3 Wells A. EGF receptor. Int J Biochem Cell Biol 1999; 31: 637–43.
4 Galizia G, Lieto E, Ferraraccio F et al. Prognostic significance of epidermal
growth factor receptor expression in colon cancer patients undergoing curative
surgery. Ann Surg Oncol 2006; 13: 823–35.
5 Jones HE, Goddard L, Gee JM et al. Insulin-like growth factor-I receptor
signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human
breast and prostate cancer cells. Endocr Relat Cancer 2004; 11: 793–814.
were inoculated subcutaneously with HCT116⁄Spry2 or HCT116⁄v cells into both flanks with 2 · 106cells⁄injection site. Mice were administered
with gefitinib (50 mg⁄kg⁄day) by oral gavage at days 1–5 and 8–12. They were sacrificed at 29 days after tumor cell inoculation or when their
tumors exceeded 17 mm in diameter. (a) Representative HCT116⁄v and HCT116⁄Spry2 tumor-bearing mice after gefitinib treatment at day 29.
(b) Representative immunostaining of Spry2 in HCT116⁄v and HCT116⁄Spry2 tumor sections (original magnification, ·100). (c) Tumor volume was
measured at indicated days from each implant. Seven of 10 implants from HCT116⁄v cells developed palpable tumors, whereas only two of 10
implants from HCT116⁄Spry2 cells formed tumor masses. HCT116⁄v tumors were significantly larger than HCT116 ⁄Spry2 tumors (P = 0.006).
Sprouty2 (Spry2) enhances the effect of gefitinib on suppressing colon cancer growth in a xenograft model. Groups of five nude mice
Feng et al.Cancer Sci |
|vol. 101|no. 9|
ª ª 2010 Japanese Cancer Association
6 Ranson M, Mansoor W, Jayson G. ZD1839 (IRESSA): a selective EGFR-TK
inhibitor. Expert Rev Anticancer Ther 2002; 2: 161–8.
7 Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor
receptor (EGFR) activation and response to gefitinib and other EGFR-
targeting drugs. Clin Cancer Res 2006; 12: 7242–51.
8 Lynch TJ, Bell DW, Sordella R et al. Activating mutations in the epidermal
growth factor receptor underlying responsiveness of non-small-cell lung
cancer to gefitinib. N Engl J Med 2004; 350: 2129–39.
9 Van Schaeybroeck S, Karaiskou-McCaul A, Kelly D et al. Epidermal growth
factor receptor activity determines response of colorectal cancer cells to
gefitinib alone and in combination with chemotherapy. Clin Cancer Res 2005;
10 Zampino MG, Magni E, Santoro L et al. Epidermal growth factor receptor
serum (sEGFR) level may predict response in patients with EGFR-positive
advanced colorectal cancer treated with gefitinib? Cancer Chemother
Pharmacol 2008; 63: 139–48.
11 Lievre A, Bachet JB, Le Corre D et al. KRAS mutation status is predictive of
response to cetuximab therapy in colorectal cancer. Cancer Res 2006; 66:
12 Lievre A, Bachet JB, Boige V et al. KRAS mutations as an independent
prognostic factor in patients with advanced colorectal cancer treated with
cetuximab. J Clin Oncol 2008; 26: 374–9.
13 Frattini M, Saletti P, Romagnani E et al. PTEN loss of expression predicts
cetuximab efficacy in metastatic colorectal cancer patients. Br J Cancer 2007;
14 Hacohen N, Kramer S, Sutherland D, Hiromi Y, Krasnow MA. Sprouty
encodes a novel antagonist of FGF signaling that patterns apical branching of
the Drosophila airways. Cell 1998; 92: 253–63.
15 Wong ES, Fong CW, Lim J et al. Sprouty2 attenuates epidermal growth factor
receptor ubiquitylation and endocytosis, and consequently enhances Ras⁄ERK
signalling. EMBO J 2002; 21: 4796–808.
16 Haglund K, Schmidt MH, Wong ES, Guy GR, Dikic I. Sprouty2 acts at the
downregulation. EMBO Rep 2005; 6: 635–41.
17 Kim HJ, Taylor LJ, Bar-Sagi D. Spatial regulation of EGFR signaling by
Sprouty2. Curr Biol 2007; 17: 455–61.
18 Lee CC, Putnam AJ, Miranti CK et al. Overexpression of sprouty 2 inhibits
HGF⁄SF-mediatedcell growth, invasion,
Oncogene 2004; 23: 5193–202.
19 Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc
Hum Genet 2007; 54 12.10.1–12.10.24.
20 Tarnawski AS, Pai R, Tanigawa T, Matysiak-Budnik T, Ahluwalia A. PTEN
silencing reverses aging-related impairment of angiogenesis in microvascular
endothelial cells. Biochem Biophys Res Commun 2010; 394: 291–6.
21 Colquhoun AJ, McHugh LA, Tulchinsky E, Kriajevska M, Mellon JK.
Combination treatment with ionising radiation and gefitinib (‘Iressa’,
ZD1839), an epidermal growth factor receptor (EGFR) inhibitor, significantly
inhibits bladder cancer cell growth in vitro and in vivo. J Radiat Res (Tokyo)
2007; 48: 351–60.
22 Tonra JR, Deevi DS, Corcoran E et al. Synergistic antitumor effects of
combined epidermal growth factor receptor and vascular endothelial growth
factor receptor-2 targeted therapy. Clin Cancer Res 2006; 12: 2197–207.
23 Mok TS, Wu YL, Thongprasert S et al. Gefitinib or carboplatin-paclitaxel in
pulmonary adenocarcinoma. N Engl J Med 2009; 361: 947–57.
24 Zhu CQ, da Cunha Santos G, Ding K et al. Role of KRAS and EGFR as
biomarkers of response to erlotinib in National Cancer Institute of
Canada Clinical Trials Group Study BR.21. J Clin Oncol 2008; 26: 4268–
25 Mackenzie MJ, Hirte HW, Glenwood G et al. A phase II trial of ZD1839
(Iressa) 750 mg per day, an oral epidermal growth factor receptor-tyrosine
kinase inhibitor, in patients with metastatic colorectal cancer. Invest New
Drugs 2005; 23: 165–70.
26 Ciardiello F, Caputo R, Bianco R et al. Antitumor effect and potentiation of
cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an
epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin
Cancer Res 2000; 6: 2053–63.
27 Paez JG, Janne PA, Lee JC et al. EGFR mutations in lung cancer: correlation
with clinical response to gefitinib therapy. Science 2004; 304: 1497–500.
28 Nagahara H, Mimori K, Ohta M et al. Somatic mutations of epidermal
growth factor receptor in colorectal carcinoma. Clin Cancer Res 2005; 11:
29 Lo TL, Yusoff P, Fong CW et al. The ras⁄mitogen-activated protein kinase
pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and
sprouty 2 are deregulated in breast cancer. Cancer Res 2004; 64: 6127–
30 Sutterluty H, Mayer CE, Setinek U et al. Down-regulation of Sprouty2 in non-
small cell lung cancer contributes to tumor malignancy via extracellular
signal-regulated kinase pathway-dependent and -independent mechanisms.
Mol Cancer Res 2007; 5: 509–20.
31 Fritzsche S, Kenzelmann M, Hoffmann MJ et al. Concomitant down-
regulation of SPRY1 and SPRY2 in prostate carcinoma. Endocr Relat Cancer
2006; 13: 839–49.
32 Fong CW, Chua MS, McKie AB et al. Sprouty 2, an inhibitor of mitogen-
activated protein kinase signaling, is down-regulated in hepatocellular
carcinoma. Cancer Res 2006; 66: 2048–58.
33 Edwin F, Singh R, Endersby R, Baker SJ, Patel TB. The tumor suppressor
PTEN is necessary for human Sprouty 2-mediated inhibition of cell
proliferation. J Biol Chem 2006; 281: 4816–22.
34 Douillard JY, Shepherd FA, Hirsh V et al. Molecular predictors of outcome
with gefitinib and docetaxel in previously treated non-small-cell lung cancer:
data from the randomized phase III INTEREST trial. J Clin Oncol 2010; 28:
35 Jackman DM, Miller VA, Cioffredi LA et al. Impact of epidermal growth
factor receptor and KRAS mutations on clinical outcomes in previously
untreated non-small cell lung cancer patients: results of an online tumor
registry of clinical trials. Clin Cancer Res 2009; 15: 5267–73.
36 Kokubo Y, Gemma A, Noro R et al. Reduction of PTEN protein and loss of
epidermal growth factor receptor gene mutation in lung cancer with natural
resistance to gefitinib (IRESSA). Br J Cancer 2005; 92: 1711–9.
37 Noro R, Gemma A, Miyanaga A et al. PTEN inactivation in lung cancer cells
and the effect of its recovery on treatment with epidermal growth factor
receptor tyrosine kinase inhibitors. Int J Oncol 2007; 31: 1157–63.
38 Chang YS, Yeh KT, Chang TJ et al. Fast simultaneous detection of K-RAS
mutations in colorectal cancer. BMC Cancer 2009; 9: 179.
39 Lin SY, Chen PH, Wang CK et al. Mutation analysis of K-ras oncogenes in
gastroenterologic cancers by the amplified created restriction sites method. Am
J Clin Pathol 1993; 100: 686–9.
Additional Supporting Information may be found in the online version of this article:
Fig. S1. K-ras mutation of colon cancer cell lines. (a) Multiplex PCR and extension analysis of mutations in K-ras codons 12 and 13 were done
in six colon cancer cell lines. A 289-bp fragment was detected in SW620, SW480, and LS174T cells, indicating the mutation of K-ras codon 12.
HCT116 cells were found to have a 109-bp fragment representing K-ras codon 13 mutation. (b) DNA auto-sequencing of K-ras confirmed that
HT29 and C2BBel cells harbored wild-type K-ras and that the remaining four cell lines had mutations in either codon 12 or 13. K-ras mutation
analysis: We used multiplex PCR and primer extension analysis of mutation in K-ras codons 12 and 13 as previously described.(38,39)For direct
sequencing of the K-ras gene, the extracted DNA was subjected to PCR amplification using forward 5¢-CCT TAT GTG TGA CAT GTT CT-3¢
and reverse 5¢-GTC CTG CAC CAG TAA TAT GC-3¢ primers. Subsequently, PCR fragments were purified using the PCR clean-up system (Pro-
mega, Madison, WI, USA) and subjected to cycle sequence reactions using BigDye Terminators (Applied Biosystems, Foster City, CA, USA).
The sequence fragments were precipitated and analyzed using an automated sequencer (ABI 3130XL; Applied Biosystems).
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ª ª 2010 Japanese Cancer Association