Download Supplementary Information (doc 2954K)

Supplementary Information for
A peptide that inhibits function of Myristoylated Alanine - Rich C Kinase
Substrate (MARCKS) reduces lung cancer metastasis
Ching-Hsien Chen, Phillip Thai, Ken Yoneda, Kenneth B. Adler,
Pan-Chyr Yang and Reen Wu
1
Supplementary Data
Figure S1
Left, comparison of the migration ability and between parental H441 cells and
H441/F3 cells derived from H441 cell line. Cells were respectively introduced a
wound/scratch and cells migration into the wound area were evaluated after 8 hours.
Right, Western blot analysis of MARCKS expression and its Ser159/163
phosphorylation in the two cell lines.
2
Figure S2
MANS and RNS peptides at 100 M level have no adverse effects on cell viability of
various lung cancer cells from CL1-5, PC9 and A549 cultures. At various times after
the treatment, trypan blue dye exclusion assay was used to count cell viability. The
cell viability was calculated by the number of viable cells/the number of total cells x
100.
3
Figure S3
Effects of MANS peptide on the suppression of CL1-5 cells metastasis in vivo.
After pretreatment with either 100 M RNS or MANS peptide for 4 hours, dissociated
CL1-5 cells were orthotopically injected to the left lobe of the mouse lung as described
in Methods. After a week, mice were injected intraperitoneally with 500 μl of PBS or
with PBS containing either the RNS or MANS peptide (50 nmoles) once every three
days thereafter for a total of 6 injections up to day 25. At day 25, mice were sacrificed
and organs were removed and examined. Top, gross (Left) and H&E staining (Right)
pictures of various organs removed from mice. The arrows indicate tumor nodules in
the organ. Scale bar: 20 μm. Bottom, quantification of the average pulmonary
metastasis nodules from mice with injected cancer cells and treated with RNS or
MANS peptide as described (*P < 0.05 versus RNS).
4
Figure S4
Effects of MANS or RNS peptide on the suppression of MARCKS and AKT
phosphorylation in CL1-0/F3 cells. Cells were treated with different amounts of MANS
or RNS peptide as indicated. Twelve hours later, cells were harvested for western blot
analysis with appropriate antibodies.
5
Supplementary Methods
Reagents and antibodies
RPMI-1640 medium, fetal bovine serum and penicillin-streptomycin was
purchased from Life Technologies Inc (Carlsbad, CA). Lipofect-AMINE™ was
purchased from Invitrogen (Carlsbad, CA). VECTASTAIN® Elite ABC Kit (Rabbit IgG),
VECTOR® Hematoxylin QS nuclear counterstain and DAB solution were purchased
from VECTOR Laboratories Inc (Burlingame, CA). TRITC-conjugated phalloidin,
12-0-tetradecanoyl phorbol-13-acetate (TPA), ROCK inhibitor (Y27632) and PKCs
inhibitor (Calphostin C) were purchased from Sigma (St Louis, MO). Anti-pSer159/163
MARCKS (clone EP2113Y), anti-MARCKS (clone EP1446Y) were purchased from
Epitomics (Burlingame, CA). Anti-E-cadherin, anti-Slug, anti-pSer473 AKT, anti-AKT,
anti-pY-p85 PI3K binding motif, anti-PI3K and anti-β-actin antibodies were purchased
from Cell Signaling Technology, Inc (Danvers, MA). MARCKS siRNAs (MARCKS
siRNA Smartpool) and DharmaFECT siRNA transfection reagents were purchased
from Dharmacon, Inc (Lafayette, CO).
Patient tumor specimens and immunohistochemical staining
Lung tumor tissue and adjacent normal tissue specimens were obtained from
patients with histologically confirmed NSCLC who underwent surgical resection at the
UC Davis Medical Center. None of the patients had received pre-operative adjuvant
chemotherapy or radiation therapy. This investigation was approved by the
Institutional Review Board of the UC Davis Health System. Written informed consent
was obtained from all patients. The post-surgical pathologic stage of each tumor was
classified according to the international TNM classification 1. Formalin-fixed and
paraffin-embedded specimens were used, and level of phospho-MARCKS was
6
analyzed by immunohistochemical staining as described previously 2. A four-point
staining intensity scoring system was devised to confirm the relative expression of
MARCKS in cancer specimens; the score ranged from zero (no expression) to 3
(highest-intensity staining). The results were classified into two groups according to
the intensity and extent of staining: in the low-expression group, either no staining was
present (staining intensity score=0) or positive staining was detected in less than 10%
of the cells (staining intensity score=1), and in the high-expression group, positive
immunostaining was present in 10-25% of the cells (staining intensity score=2) or in
more than 25% of the cells (staining intensity score=3). These results were also
reviewed and scored independently by two pathologists.
In vivo subcutaneous and orthotopic implantation
Six-week-old nude mice and NOD severe combined immunodeficiency (SCID)
mice (supplied by Charles River Laboratories) were housed four mice per cage and
fed autoclaved food ad libitum. For subcutaneous xenografts assay, the dorsal region
of nude mice was injected subcutaneously with 2  106 PC9 cells. Injected mice were
housed for 32 days and examined every 5 days for tumors. Groups randomized and
treatment started when tumor size was 3–5 mm (the tumor volume was calculated by
using the formula V=0.4Xab2, where a and b are the longest and shortest diameters of
the tumors, respectively). Each experimental group included at least 6 mice bearing
tumors. These nude mice bearing subcutaneous tumors were treated for 21 days with
intratumoral injections of PBS (Con), RNS, or MANS peptide at the dosage of 50
nmoles every two days. After 32 days of tumor of implantation, these mice were
sacrificed and the lungs of these mice were removed, weighed and fixed in 10%
formalin. Embedded tissues were sliced into 4 m sections which were stained with
H&E staining and the number of lung micrometastatic lesions was counted under a
7
dissecting microscope. For orthotopic implantation, the protocol is as described
previously3-4. Briefly, PC9 or CL1-5 cells (5  104 cells in 40 l PBS containing 10 ng
Matrigel) were inoculated into the left lung of mice by insulin syringe with 29-G needle.
After 7 day of implantation, the mice were subjected to systemic treatment with either
PBS only (Con), RNS, or MANS peptide at 50 nmoles by intraperitoneal injections
every three days. The mice were sacrificed 25 days after implantation. The numbers
of lung tumor colonies from orthotopic implanted mice were evaluated by gross and
microscopic examination. Mouse experiments were approved by the Institutional
Animal Care and Use Committee of UC Davis.
8
Supplementary References
1 Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest
1997; 111:1710-1717.
2 Fan T, Li R, Todd NW et al. Up-regulation of 14-3-3zeta in lung cancer and its
implication as prognostic and therapeutic target. Cancer Res 2007; 67:7901-7906.
3 Chen CH, Chuang SM, Yang MF, Liao JW, Yu SL, Chen JJ. A novel function of
YWHAZ/beta-catenin axis in promoting epithelial-mesenchymal transition and lung
cancer metastasis. Mol Cancer Res 2012; 10:1319-1331.
4 Pan SH, Chao YC, Hung PF et al. The ability of LCRMP-1 to promote cancer
invasion by enhancing filopodia formation is antagonized by CRMP-1. J Clin Invest
2011; 121:3189-3205.
9