Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target.

stable across colon cancer tissue samples, these reference genes are not suitable as an internal standard for comparison of gene expression between normal colon tissues and colon cancers.
Sequence of the tagged constructs was confirmed by sequencing both strands (Cleveland Genomics, Cleveland, OH).

Transfection and detection of CEMIP from cell lysates and cell media. SW480 and
VACO-400 cells were transfected with either T7-or V5/His-tagged CEMIP expression vector using Fugene (Roche, Indianapolis, IN) for SW480 or Effectene (Qiagen, Valencia, CA) for VACO-400 following the manufacturer's protocols. Seventy-two hours after transfection, the media was removed and clarified by centrifugation for 5 min at 2,000 rpm and the supernatant was collected in a fresh tube. Epitope-tagged CEMIP was immunoprecipitated from media samples using either a 1:1000 dilution of mouse, anti-T7 antibody (Novagen, #69522-3) or a 1:333 dilution of mouse, anti-V5 antibody (Invitrogen, #46-1157) and rocking overnight at 4°C. The next day Protein G beads (Upstate Biotechnology, #16-266) were added to each sample and rocked at 4°C for 1.5 h. The samples were then washed 3 times with RIPA buffer (1x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) containing Complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN) and loaded onto a 4-12% Bis-Tris SDS-PAGE (Invitrogen, Carlsbad, CA) for protein separation. The accompanying pellet of transfected cells were similarly lysed for 15 min at 4°C in RIPA buffer containing protease inhibitor cocktail. Lysates were then centrifuged for 10 min at 14,000 rpm at 4°C to remove the insoluble fraction, and the clarified supernatants were also loaded onto the SDS-PAGE gel. Equal percentage amounts of total cell lysates and total cell culture media were represented on each SDS-PAGE gel. Proteins were transferred onto Immobilon™-P PVDF membranes (Millipore, Billerica, MA). Membranes were blocked with 5% nonfat milk, probed with either a 1:3000 dilution of mouse, anti-V5 antibody, or a 1:3000 dilution of mouse, anti-T7 antibody, and developed by using a 1:1500 dilution of donkey anti-mouse horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., #715-035-150). Enhanced Chemiluminescence Plus (Amersham Biosciences, Piscataway, NJ) and a STORM 840 phosphoimager were used to detect protein bands.

Creation of a HeLa cell clone that stably secretes a V5-His epitope tagged CEMIP protein.
To purify and characterize the properties of CEMIP protein, we developed a HeLa cell line (clone A11-4) that expresses a compound V5-His epitope-tagged CEMIP under the inducible control of a doxycycline regulated promoter. V5/His-tagged CEMIP was cut out of the pcDNA3.1/V5/His vector with KpnI and PmeI, the overhangs filled in, and blunt-end ligated into the PmeI site of pcDNA4/TO/myc-His (Invitrogen, Carlsbad, CA). T-REx™-HeLa cells were seeded at 12,000 cells/100 mm dish. The next day the cells were transfected with 20 µg of pcDNA4/CEMIP-V5/His plasmid construct using TransIT ® -HeLa Monster transfection reagent (Mirus, Madison, WI) following the manufacturer's protocol. Two days after transfection, pcDNA4-containing cells were selected for using 150 µg/ml of zeocin. Stable clones that grew out of the zeocin selection were confirmed for inducible CEMIP expression by treatment with 1 µg/ml doxycycline for 24 h and then assayed by Western blot. The procedure for creating a pool of stable control clones was the same as above except an empty pcDNA4/TO/myc-His vector was used for transfection.
Purification of recombinant CEMIP. T-REx™-HeLa cells expressing the inducible V5/Histagged CEMIP were seeded into 1700 cm 2 expanded surface roller bottles (Corning Inc., Corning, NY) and grown to ~75% confluence, whereupon fresh media was added that contained 1 µg/ml doxycycline and low IgG serum. After 72 h, the media was collected and centrifuged for 5 min at 2,000 rpm to remove any cellular debris. The media was then frozen and sent to Roche Protein Expression Group (RPEG) (Roche, Indianapolis, IN) for purification of the V5/His-tagged CEMIP.
Briefly, CEMIP was purified by a two-step process which comprised of a Ni-affinity step to bind the 6xHis tag of CEMIP and then a size exclusion chromatography step using a Sephadex G-200 column.
Aliquots of the collected fractions were run on an SDS-PAGE gel and analyzed by Coomassie blue staining. The CEMIP-containing fractions were pooled and a concentrated using a Centricon YM-10 centrifugal filter (Millipore, Billerica, MA), and the protein concentration was determined using the Bradford assay. To determine the purity of the CEMIP protein, a sample was run on an SDS-PAGE gel and densitometry was performed using an AlphaImager (Alpha Innotech, San Leandro, CA).
Identity of the CEMIP band was confirmed both by mass spectrometry and by western blotting with an anti-6xHis-tag antibody (Roche, #11922416001).

Generation of anti-CEMIP monoclonal antibodies. Generation of anti-CEMIP monoclonal
antibodies was performed under contract by Celliance Corporation (Norcross, GA). Briefly, 25 µg of purified recombinant protein was added to 200µl of Ribi or Complete Freund's Adjuvant and then injected sub-cutaneously into female Balb/c mice. Supernatants from the hybridomas were first screened for anti-CEMIP activity by ELISA using purified V5/His-tagged CEMIP. Medias positive by ELISA for anti-CEMIP activity were then further screened for endogenous CEMIP western blot activity using purified T7-tagged CEMIP and FET cell lysates, as well as screened for immunoprecipitation activity using media collected from CEMIP expressing (FET and V411) and non-expressing (V364 and RKO) cell lines. Hybridomas that tested positive for anti-CEMIP activity were injected into mice and the monoclonal antibodies were purified from the ascites using Protein G beads from Roche following the manufacture's protocol.