Online Data Supplementfor

Up-regulation of MARCKS in Kidney Cancer and its Potential as a Therapeutic Target

Ching-Hsien Chen*, Lon Wolf R. Fong, Eric Yu, Reen Wu, Josephine F. Trott,

Robert H. Weiss*

Supplementary Methods

Reagents and antibodies

Dulbecco's Modified Eagle's medium, RPMI-1640 medium,fetal bovine serum and penicillin-streptomycin were 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 andDAB solutionwerepurchased from VECTOR LaboratoriesInc. (Burlingame, CA). Anti-CD 31, anti-pSer158 MARCKS (clone EP2113Y) and anti-MARCKS (clone EP1446Y) werepurchased from Abcam (Cambridge, MA).Anti-pSer159/163 MARCKS (clone D13D2),anti-pSer473 AKT, anti-AKT, anti-pSer2448 mTOR,anti-mTOR,anti-cleaved caspase 3, anti-PCNA and anti-β-actin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Plasmid constructs and primers

For generation of MARCKS shRNAplasmids, the oligonucleotide of shRNAs(shRNA: 5'-GAGCGCTTCTCCTTCAAGAA-3' and its complementary strand: 5'-TTCTTGAAGGAGAAGCGCTC-3') were synthesized, annealed and cloned into the pGreenPuroshRNA expression lentivector (System Biosciences, Mountain View, CA). For identification and cloning of the MARCKSfull-length cDNA, total RNA was isolated from cancer cells using Trizol reagent (Life Technologies, Carlsbad, CA). First-strand cDNA was reverse-transcribed with SuperScript II reverse transcriptase and oligo-dT primer(Life Technologies, Carlsbad, CA). The MARCKS coding region was amplified by polymerase chain reaction (PCR) using the forward primer: 5’-GGATCCATGGGTGCCCAGTTCTCCAAGACCGCAGC-3’, which introduced a BamHI site, and the reverse primer: 5’-TCTAGACTCTCTGCCGCCT CCGCTGGGGGGGCT-3’, which introduced an XbaI site. The amplified product was cloned intothe pcDNA3.1 vector (Invitrogen, Carlsbad, CA) and sequenced to ensure that no mutations were introduced during the PCR amplification.The all primers for quantitative real-time PCR used were as follows: the VEGFAforward primer5'-CCTTGCTGCTCTACCTCCA-3'andthe reverse primer5'-CAAATGCTTTCTCCGCTCT-3';the IL-6forward primer5'-TGACAAACAAATTCGGTACATCCT-3'andthe reverse primer5'-AGTGCCTCTTTGCTGCTTTCAC-3'; the MMP9forward primer5'-GGGACGCAGACATCGTCATC-3'andthe reverse primer5'-TCGTCATCGTCGAAATGGGC-3'; the Cox-2forward primer5’-ATCATTCACCAGGCAAATTGC-3’andthe reverse primer5’-GGCTTCAGCATAAAGCGTTTG-3'; the MARCKSforward primer5-TTGTTGAAGAAGCCAGCATGGGTG-3andthe reverse primer5-TTACCTTCACGTGGCCATTCTCCT-3.

Patient tumor specimens and immunohistochemical staining

Kidneytumors (which include 56 patients) were obtained from patients with histologically confirmed RCC who underwent surgical resection at theUC DavisComprehensive Cancer Center(Sacramento, CA). This investigation was approved by the Institutional Review Board of the UC Davis Health System. Written informed consent was obtained from all patients.Formalin-fixed and paraffin-embedded specimens were used, and immunohistochemical staining was performed for phospho-MARCKS levels as well as MARCKS expression. Detailed experimental procedures were modified from the paraffin immunohistochemistry protocol supplied by the manufacturer (Cell Signaling, Danvers, MA).Theslides were de-paraffinized in xylene and rehydrated in graded alcohol and water. An antigen retrieval step (10 nMsodium citrate (pH 6.0) at a sub-boiling temperature) was used for each primary antibody. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide followed by blocking serum and incubation with appropriate antibodies overnight at 4°C. Detection of immunostaining was carried out by using the VECTASTAIN®ABC system, according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). A four-point staining intensity scoring system was devised to confirm the relative expression of MARCKS and phospho-MARCKS in cancer specimens; scores ranged from zero (no expression) to 3 (highest-intensity staining) as described previously1-4. The results were classified into two groups according to the intensity and extent of staining: in the low-expression group,staining was observed in 0–1% of the cells (staining intensityscore = 0),in less than 10% of the cells (staining intensity score =1), or in 10%-25% of the cells (stainingintensity score = 2); in thehigh-expression group, staining was presentmore than 25% of the cells (staining intensity score = 3).

Xenograft models of kidney cancer

Animal usage protocols were periodically reviewed and approved by Institutional Animal Care and Use Committee at UC Davis. Six-week-old nude mice (supplied by Charles River Laboratories, San Diego, CA)were housed four mice per cage and fed autoclaved food ad libitum. We have shown that Caki-1 and 786-O cells exhibited higher MARCKS expression and phospho-MARCKS levels (Figure 2a), thus they are suitable cell lines for generating stable MARCKS-knockdown condition by shRNA approaches and used fora subcutaneous xenograft model. The dorsal region of nude mice wasinjected subcutaneously with 5106 MARCKS shRNA-silencedcells or shRNA control cells(MARCKS-manipulated cells) and these mice were examined every 3 days for tumor size.The tumor volume was calculated by using the formula V=0.5Xab2, where a and b are the longest and shortest diameters of the tumors, respectively. After 28 days, the xenografted tumors were removed, weighed and fixed in 10% formalin; embedded tissues were sliced into 4 m sections, which were stained with hematoxylin-eosin and indicatedantibodies for histological analysis.

Quantitative PCR

The mRNA expression level of target genes wasdetected by real-time reverse transcription polymerase chain reaction (RT-qPCR) usingprimers as described in theSupplementary Methods. The house keeping gene TATA-box binding protein (TBP) was used as the reference gene. The relative expression level of target genes compared with that of TBP was defined as –CT = –[CTtarget–CTTBP]. The target/TBP mRNA ratio was calculated as 2 –CT K, where K is a constant.

Immunoblotting

Immunoblotting has been previously described27. Briefly, whole cell lysates were prepared by lysing cells in a lysis buffer (50 mMTris-HCl (pH 7.4), 1% NP-40, 150 mMNaCl, 1 mM EDTA, 20 μg/ml leupeptin, 1 mM PMSF and 20 μg/ml aprotinin), and proteins were then separated by SDS-PAGE. Immunoblotting was conducted with appropriate antibodies followed by chemiluminescent detection.

Cell viability, proliferation and colony formation assays

Trypanblueexclusiontest, MTS proliferation and thiazolyl blue tetrazolium bromide (MTT)assays were used to quantitate viable cell numbers. For the Trypanbluetest, cellswere plated on 12-well plates and treated with the indicated agents. After 48 hours,both attached and detached cells were collected andthen stained with 0.2%trypanblue(0.1% final concentration), and the number of trypan blue-positive and -negative cells was counted using a haemocytometer under low-power microscopy. For MTS and MTT assays, cells were seeded into 96-well plates and cultured for the indicated treatment.Cell proliferationand viability were evaluated by MTSand MTT assays according to the manufacturer’s protocols(MTS:Promega, Madison, WI and MTT: Chemicon, Temecula, CA, respectively).The absorbance measuredwas on a multi-well scanning spectrophotometer (Victor3; Perkin-Elmer, Boston, MA).For the anchorage-dependent growth assay, 200 cells were seeded in each well of6-well plates.MARCKS-manipulated cells were cultured in complete culture medium for 7 days.Caki-1, 786-O and A498 cellswere treated with the MPS peptideat the indicated concentrations for 4 days and then changed to the complete culture medium; these cells were further incubated for 5 days. Colonies were stained using 0.001% crystal violet and the number of colonies with a diameter greater than 0.5 mm was counted under an inverted microscope.

Transwell migrationassay

An in vitro cell migration assay was performed as previously described 5using transwell chambers (8-μm pore size; Costar, Cambridge, MA). Briefly, 5x103cells were seeded on top of the polycarbonate filters, and 0.5 ml of growth mediumwas added to both the upper and lower wells. After incubation for 12 hours, filters were swabbed with a cotton swab, fixed with methanol, and then stained with Giemsa solution(Sigma-Aldrich, St Louis, MO).The cells attached to the lower surface of the filter were counted under a light microscope (10X magnification).

Peptide synthesis

TheMPS, control and scrambled peptides (95% pure) were purchased from EZBiolab Inc. (Carmel, IN). The MPS peptide consisted of amino acids 151 to 175 from the wild-type protein, KKKKKRFSFKKSFKLSGFSFKKNKK; the control peptidehad a sequence KKKKKRFDFKKDFKLDGFDFKKNKK, and the scrambled peptidehad a sequenceKRFLSKKKNKSFFGKSKKFKKKKSF.Peptides were reconstituted in phosphate-buffered saline, yielding stock concentrations of 10 mM. Stock solutions were stored at -20°C and diluted to desired concentrations on the day of the experiment.

Kinetic assay

Real-time binding of the peptide mimicking the phosphorylation site domain of MARCKS (MPS peptide, amino acids 151 to 175 from the wild-type MARCKS protein) to biotin-labeled PIP2 was evaluated using biolayerinterferometry (BLI) on an Octet RED96 system (ForteBio) following the manufacturer's instructions. Briefly, the ligand, PIP2 labeled with biotin at the sn-1 position (1000 nM in ddH2O), was immobilized on Super Streptavidin (SSA) biosensors for 10 minutes. A binding assay was performed with the MPS analyte at various concentrations from 0 to 1000 nM in ddH2O. Association and dissociation were monitored for 5 minutes. Assays were performed at 24°C. Data were analyzed using Octet Data Analysis Software 7.0 (ForteBio).

PI(3,4,5)P3 quantitation

Cells were harvested and precipitated by trichloroacetic acid. PIP3 lipids wereextracted twice from the trichloroacetic acid precipitated fraction by methanol:chloroform (2:1). Afteracidification, organic-phase lipids were used for PIP3 quantitation, basedon the protocol for the PIP3 Mass ELISA kit(Echelon Biosciences, Salt Lake, UT). Briefly, the lipid extract fromcultured cells was mixed with the PIP3-specific detector protein,which was then incubated in a PIP3-coated mircroplate for competitivebinding. After several washes, the microplate was then incubated with aHRP-linked secondary detector and tetramethylbenzidine substrate forcolor development. To stop further color development 2M H2SO4 was added. Microplates were read at a wavelength of 450 nm.A series of different dilutions of PIP3 standards were used for establishinga standard curve for each reaction. Cellular PIP3 amounts couldbe estimated by comparing the absorbance in the wells with the values inthe standard curve. Experiments were conducted in triplicate dishes andrepeated in several independent cultures with cell density7-8×106 cells/100-mm dish.

Evaluation of therapeutic interactions

The therapeutic interactions between themultikinaseinhibitor regorafenib and MPS peptide were analyzed accordingto the method of Chou and Talalay6with the help ofthe Calcusyn software suite (Biosoft, Cambridge, UnitedKingdom). Combinationindex (CI) values were determined bygenerating dose-response curves for various concentrations of regorafenib (0.0625-1 μM) treatment in conjunction with MPS peptide (6.25-100 μM) and calculations were performed at differentdrug concentrations. According to Chou and Talalay, a CI of <1 indicates a synergistic interaction, a CI of 1 indicates anadditive interaction, and a CI of >1 indicates an antagonisticinteraction6.

Supplementary Data

Table S1. Summary of clinicopathologicfeatures according to MARCKS and phospho-MARCKS expression.

Characteristic / High
No. of Patients (%) / Low
No. of Patients (%) / p-value
MARCKS
Number of patients / n=28 / n=28
Age (meanSD) / 6215 / 5811 / 0.27†
Gender
Male / 14 (25) / 19 (34) / 0.28‡
Female / 14 (25) / 9 (16)
Grade* / 0.04‡
G1 / 6 (11) / 11 (21)
G2
G3
G4 / 9 (17)
11 (21)
0 (0) / 12 (22)
3 (6)
1 (2)
phospho-MARCKS
Number of patients / n=37 / n=19
Age (meanSD) / 5813 / 6413 / 0.11†
Gender
Male / 19 (34) / 14 (25) / 0.28‡
Female / 18 (32) / 5 (9)
Grade* / 0.12‡
G1 / 8 (15) / 9 (17)
G2
G3
G4 / 15 (28)
11 (21)
0 (0) / 6 (11)
3 (6)
1 (2)
†T test.
‡Fisher’s exact test.

*Some patients without grade information

Table S2.Numberof patientswith either positive or negative nuclear staining forphospho-MARCKSaccording to tumor grade.

Grade
G1 G2 G3 G4 p-value
phospho-MARCKS
Nuclear-positive / 11 13 / 10 1
Nuclear-negative / 6 8 / 4 0
Fisher’s exact test 0.916

*note: n=53 (Some patients without grade information).

Supplementary Figure Legends

Supplementary Figure S1.Related to Figure 1

(a) Functional interactions between HIF1A and MARCKS. Genome-scale integrated analysis of gene networks in kidney tumors ((b) Normalized expression of MARCKS inclear cell renal cell carcinoma tissues (n=534) versus normal kidney tissues (n=72) using the TCGA dataset.p < 0.01, t = 12.98, dof = 604 (two-tailed, unpaired t-test).

Supplementary Figure S2. Related to Figure 2

(a)Effects of ectopic V5-tagged wild-type or PSD-mutated (S159/163A) MARCKS expression on cell proliferation (top) and MARCKS phosphorylation(bottom) innormal human kidney epithelial cells.NHKcells were transfected with either wild type or mutant (S159/163A) V5-tagged MARCKS. After 24 hours of transfection, cells were subjected to MTS assays (top)and Western blot analysis (bottom). (b)Effects of MARCKS-knockdown on cell growthas determinedby colony formation assays. Colonies were stained and visualized microscopically as described in Materials and Methods.A representative view of each cell lineis shown in the upper panel. Bottom, colonies were counted in a blinded fashion and quantification. (c) Reduced cell spreading and motility in MARCKS-knockdown RCC cells. Caki-1 cellswere transductedwithcopGFP-taggedMARCKS-specific shRNA or control non-specific shRNAas indicated. After 72 hours of transduction, cells were subjected to microscopic examination (left)and transwell migration (right)assays.Left, cell morphology (top) and transductionefficiency (bottom); green: cells transducted with pGreenPuroshRNA expression lentivector. Right, decreased cell motility in MARCKS-knockdown cells.Data expressed as mean ± SD (n = 3), *p < 0.05 as compared to cells receiving control shRNA.(d)MPS peptide treatment inhibited MARCKS phosphorylation of RCC cells. 786-O (left), Caki-1 (middle) and A498 (right) cells were treated with either 100 µMcontrol peptide or 100 µMMPS peptidefor 48 hours and thensubjected to Western blot analysis.(e)Effect of MPS peptide on the viability of various RCC cell lines and normal human kidney epithelial cells. Cells were exposed to the indicated peptides (200 µM)including MPSpeptide anda controlor scrambled peptide.After 4 days of peptide treatment,the number of viable cells was visualized by using crystal violet staining. A representative example of three independent experiments is shown.

Supplementary Figure S3. Related to Figure 3

(a) Immunohistochemical staining of PCNA and activated caspase-3 (c-caspase-3) in 786-O xenografted tumorsas described inFigure 3 c-d. A representativeimage is shown.

Supplementary Figure S4.Related to Figure 4

PANELa-d: 786-O (a), A498(b), Caki-1 (c) and ACHN(d) cellswere infected with MARCKS-specific or non-specific shRNA-containing lentiviruses. After 72 hours of infection, expression of HIF-target genes was determined by real-time RT-qPCR (n=3, *p0.05 versuscontrol shRNA).(e)Biolayerinterferometryanalysis of the bindingof the peptides from the MARCKS phosphorylation site domain (PSD) to biotin-labeled PIP2.(f)PIP3 levels in genetically modified RCC cells. *p < 0.05 versus control shRNA (mean ± SD, 786-O: n=4; Caki-1: n=3).

Supplementary Figure S5.Related to Figure 6

PANELa-b: Caki-1 (a) and 786-O (b) cells were individually treated with 0.5 µM regorafenib, 50 µM MPS peptide or combinations of 0.5 µM regorafenib and 50 µM MPS peptide. Cell morphologywas photographedafter 48 hours of treatment and a representativeimage is shown.

SupplementaryReferences

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4 Chen CH, Cheng CT, Yuan Y et al. Elevated MARCKS phosphorylation contributes to unresponsiveness of breast cancer to paclitaxel treatment. Oncotarget 2015; 6:15194-15208.

5 Chu YW, Yang PC, Yang SC et al. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997; 17:353-360.

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