IGFBP-rP1, a potential molecule associated with colon cancer differentiation
- Wenjing Ruan†1,
- Shuzhen Zhu†1,
- Haibing Wang1,
- Fangying Xu1,
- Hong Deng1,
- Yu Ma1 and
- Maode Lai1Email author
© Ruan et al; licensee BioMed Central Ltd. 2010
Received: 10 June 2010
Accepted: 26 October 2010
Published: 26 October 2010
In our previous studies, we have demonstrated that insulin-like growth factor binding protein-related protein1 (IGFBP-rP1) played its potential tumor suppressor role in colon cancer cells through apoptosis and senescence induction. In this study, we will further uncover the role of IGFBP-rP1 in colon cancer differentiation and a possible mechanism by revealing responsible genes.
In normal colon epithelium, immunohistochemistry staining detected a gradient IGFBP-rP1 expression along the axis of the crypt. IGFBP-rP1 strongly expressed in the differentiated cells at the surface of the colon epithelium, while weakly expressed at the crypt base. In colon cancer tissues, the expression of IGFBP-rP1 correlated positively with the differentiation status. IGFBP-rP1 strongly expressed in low grade colorectal carcinoma and weakly expressed in high grade colorectal carcinoma. In vitro, transfection of PcDNA3.1(IGFBP-rP1) into RKO, SW620 and CW2 cells induced a more pronounced anterior-posterior polarity morphology, accompanied by upregulation with alkaline phosphatase (AKP) activity. Upregulation of carcino-embryonic antigen (CEA) was also observed in SW620 and CW2 transfectants. The addition of IGFBP-rP1 protein into the medium could mimic most but not all effects of IGFBP-rP1 cDNA transfection. Seventy-eight reproducibly differentially expressed genes were detected in PcDNA3.1(IGFBP-rP1)-RKO transfectants, using Affymetrix 133 plus 2.0 expression chip platform. Directed Acyclic Graph (DAG) of the enriched GO categories demonstrated that differential expression of the enzyme regulator activity genes together with cytoskeleton and actin binding genes were significant. IGFBP-rP1 could upreguate Transgelin (TAGLN), downregulate SRY (sex determining region Y)-box 9(campomelic dysplasia, autosomal sex-reversal) (SOX9), insulin receptor substrate 1(IRS1), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B), amphiregulin(schwannoma-derived growth factor) (AREG) and immediate early response 5-like(IER5L) in RKO, SW620 and CW2 colon cancer cells, verified by Real time Reverse Transcription Polymerase Chain Reaction (rtRT-PCR). During sodium butyrate-induced Caco2 cell differentiation, IGFBP-rP1 was upregulated and the expression showed significant correlation with the AKP activity. The downregulation of IRS1 and SOX9 were also induced by sodium butyrate.
IGFBP-rP1 was a potential key molecule associated with colon cancer differentiation. Downregulation of IRS1 and SOX9 may the possible key downstream genes involved in the process.
Insulin-like growth factor binding proteins (IGFBPs), described as essential modulators of IGF bioavailability, are a family of homologous proteins produced by many different tissues. IGFBPs have different molecular weight, amino acid composition, binding properties and distribution in biological fluids [1, 2]. The classified IGFBPs, including IGFBP1-6, cooperate in regulating signals from insulin receptors and IGF receptors. The last few years have brought complexity, but also new vistas of insights into the IGFBPs superfamily with discovery of new IGFBPs(IGFBP7-15), who exhibit a low affinity for IGF. These IGFBPs were reclassified as IGFBP-related proteins (IGFBP-rPs), whose roles in intracellular signaling, cell growth and cell metabolism are emerging [3, 4].
IGFBP-rP1 has been independently cloned in several cellular systems, and therefore has been previously identified as IGFBP7, meningioma associated cDNA 25 (mac25) [5, 6], tumor-derived adhesion factor(TAF) , and prostacylin-stimulating factor(PSF) . IGFBP-rP1 was cloned as a gene that downregulated in meningioma cell lines compared to primary cultures of benign leptomeningeal cells and as a senescence-associated gene from human mammary epithelial cells . IGFBP-rP1 is particularly intriguing due to its implicated role in cancer. In vivo, different expression patterns of IGFBP-rP1 were found in various tumor types. Upregulated expression of IGFBP-rP1 was observed in acute lymphoblasma leukemia and in thyroid cancer[9, 10]. Downregulated expression of IGFBP-rP1 was common in liver cancer, lung cancer and in meningiomas[11–13]. While both up- and downregulation of IGFBP-rP1 have been reported in breast and prostate cancer[14–17]. These findings make the role of IGFBP-rP1 complicated. In 1999, our laboratory purified the cDNA fragments of IGFBP-rP1 from colonic adenocarcinoma and normal mucosa cDNA subtraction libraries by suppressive subtractive hybridization (SSH) . Our group presented evidence that methylation of exon 1 was the key regulatory mechanism silencing the expression of IGFBP-rP1 in colon cancer cell lines. IGFBP-rP1 suppressed the proliferation, decreased the colony formation ability, and induced apoptosis and senescence in colorectal cancer cell lines [20, 21]. The expression of IGFBP-rP1 was correlated with favourable prognosis in colon cancer patients. All these findings strongly supported that IGFBP-rP1 played a potential tumor suppressor role against colorectal carcinogenesis. The tumor suppressive role of IGFBP-rP1 was also found in other types of cancer, including cervical cancer , osteosarcoma [22, 23], prostate cancer [14, 24], breast cancer , lung cancer, melanoma and thyroid cancer.
The balance among proliferation, differentiation, senescence and apoptosis is tightly regulated to maintain homeostasis of colon epithelium. Neoplastic transformation arises from multiple defects in these processes. Malignant transformation is often characterized by both deregulated cell cycle, increased cell survival and loss of differentiation [27, 28]. However, the role of IGFBP-rP1 in the differentiation in colon cancer cells remains elusive. The objectives of the present work were to uncover the role of IGFBP-rP1 in the differentiation of colon cancer and its possible responsible genes.
Materials and methods
Dulbecco's Modified Eagle's Medium (DMEM) was purchased from GIBCO Laboratories (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT, USA). Polyfect transfection reagent and RNeasy mini kit was purchased from QIAGEN (Hilden, Germany). G418 was purchased from Merck (Darmstadt, Germany). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). The polyclonal antibody of IGFBP-rP1 and of Actin were purchased from Santa Cruz Biologicals (CA, USA). The monoclonal antibody of IGFBP-rP1 and the recombinant IGFBP-rP1 protein were purchased from RD (Minneapolis, MN USA). The monoclonal antibody of carcinoembryonic antigen (CEA) and polyclonal antibody of caudal-related homeodomain transcription 2 (CDX2) were purchased from Cell Signaling (Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Zymed (San Francisco, CA, USA). Sodium Butyrate was purchased from Sigma Chemical Company (St. Louis, MO, USA). An alkaline phosphatase (AKP) kit was purchased from Pointe Scientific Inc.(Canton, MI, USA).
All 221 patients with colorectal carcinoma, 121 males and 100 females, were inhabitants of Xiaoshan District, Zhejiang Province, China. The age range of the patients was from 26 to 85 years (median, 59 years). All archival paraffin-embedded tissue blocks were collected from the Department of Pathology, Zhejiang University, and the People's No. 1 Hospital of Xiaoshan, and the Zhejiang Cancer Hospital from January 1990 to December 2000. Patient and treatment data were collected from patient records. The 221 patients had not received chemotherapy or radiotherapy prior to surgery. Paraffin-embedded tissue blocks were processed according to standard histologic procedures and stained with H&E. The type of histology: tubular adenocarcinoma (n = 164), papillary adenocarcinoma (n = 26), mucinous adenocarcinoma (n = 27), ring cell carcinoma, and undifferentiated carcinoma (n = 4), assessed by two experienced pathologists.
The immunohistochemical staining was carried out as described. Paraffin-embedded sections (5 μm thick) were first dewaxed in xylene and rehydrated with a graded ethanol series. Endogenous peroxidase was quenched by incubation in 3% H2O2 for 10 min at room temperature. Nonspecific binding was blocked by incubation in a 1:10 dilution of rabbit serum for 30 min at room temperature. Then, sections were incubated at 4°C overnight with a 1:200 dilution of goat polyclonal antibody against human IGFBP-rP1. After several washes in PBS, the sections were incubated with a 1:200 dilution of biotinylated rabbit IgG at room temperature for 30 min. Then, the slides were incubated in a 1:200 dilution of rabbit horseradish peroxidase at room temperature for 20 min. The peroxidase activity was visualized by incubating in 0.06% 3-3'-diaminobenzidine-H2O2. The sections were finally counter stained with hematoxylin. Immunohistochemical scores of each section were independently scored by two pathologists.
Human colorectal carcinoma RKO, SW620, CW2 and Caco2 cell lines were maintained in DMEM supplemented with 10% FBS in a 37°C/5% CO2 atmosphere. PcDNA3.1(IGFBP-rP1)-RKO, PcDNA3.1(IGFBP-rP1)-SW620, PcDNA3.1(IGFBP-rP1)-CW2 transfectants and the empty vector transfectants were established as previously described [20, 21], maintained in the same culture medium containing 200 μg/ml G418.
Recombinant IGFBP-rP1 stimulation assay
RKO, SW620 and CW2 cells were seeded into 6-well plate at 2 × 105 cells/plate, 4 × 105 cells/plate, 4 × 105 cells/plate, respectively. After attachment for 24 hr, the cells were replaced by free culture medium with recombinant IGFBP-rP1 protein (For RKO cells, 10 μg/ml, For SW620 and CW2 cells, 4 μg/ml). Forty-eight hours after IGFBP-rP1 addition, cell morphology was observed and photographed. Expression of differentiation markers were performed on the cell lysates and supernatants.
AKP activity assays
Cell lysates and the supernatants of the PcDNA3.1(IGFBP-rP1) transfectants, PcDNA3.1 transfectants, parental cells, and cells stimulated with recombinant IGFBP-rP1 protein were harvested as discussed above. AKP activity was determined according to the protocol of the kit. P-nitrophenyl phosphate disodium hexahydrate was used as a substrate. Synthetic alkaline phosphatase was used to construct a standard dilution curve.
Antibodies directed against actin (1:5000), CEA (1:1000), CDX2 (1:1000) were used in Western blot analyses. Cell lysates (50 μg) were resolved in prepoured Tris-glycine SDS gels (Bio-Rad, Richmond CA), and transferred to a nitrocellulose membrane (Bio-Rad). Blots were blocked in 5% nonfat milk in TBST (TBS buffer containing 0.1% tween), incubated with the primary antibody overnight at 4°C, washed in TBST, and then incubated with appropriate secondary antibody for 1 hr at room temperature. Antibody binding was detected using enhanced chemiluminescence reagent according to the manufacturer's instructions.
RNA isolation and microarray hybridization
We applied Affymetrix HG133 plus 2.0 chip to detect differentially expressed genes transfected by PcDNA3.1(IGFBP-rP1) in RKO cells. As technical replication of Affymetrix chip were more than 99% consistent, we performed biological replications to reduce sampling variability. We selected three single-cell clones of PcDNA3.1(IGFBP-rP1) transfectants, identified as RP5, RP6, RP7, also three control vector transfectants as control, named as EV5, EV6, EV7. Cells were divided into three paired groups, RP5 VS EV5, RP6 VS EV6, RP7 VS EV7. The paired cells were cultured in the same condition and harvested at the same time to minimize gene expression changes due to cell culture conditions. Total RNA was isolated from cells using RNeasy mini kit (Qiagen, Germany). RNA was quantitated by UV absorbance at 260 and 280 nm and assessed qualitatively using an RNA LabChip and Bioanalyzer 2100 (Agilent, Palo Alto, CA). To generate a biotinylated probe, cDNA was synthesized (Superscript cDNA synthesis kit, Invitrogen, San Diego, CA) from 4 μg RNA using an oligo(dT) primer with a T7 RNA polymerase promoter at the 5' end. The cDNA was made double stranded and used in an in vitro transcription reaction (Enzo Diagnostics) with T7 RNA polymerase to synthesize biotinylated product for hybridization to Affymetrix GeneChips HU133 plus 2.0 using the Affymetrix recommended protocolhttp://www.affymetrix.com/support/technical/manual/expression_manual.affx. Human HG-U133 plus 2.0 chips (Affymetrix, Inc.) were hybridized with 15 μg of fragmented labeled cRNA overnight at 45°C, washed (Genechip Fluidics Station 400; Affymetrix), and scanned (GeneArray Scanner; Affymetrix) according to Affymetrix protocols. Together 6 chips were performed.
Microarray data analysis
Scanned images of microarray chips were analysed by the GeneChip Operating Software (GCOS1.2) from Affymetrix. The total HG-U133 Plus 2 signal was normalized to an arbitrary signal intensity value of 500. Differentially expressed genes between IGFBP-rP1 transfectants and the controls in the groups were identified using the GCOS change algorithm and Rank Products (RP) following RMA (Robust Multiarray Analysis, One-sided Wilcoxon's Signed Rank Test). A total of three possible pairwise comparisons were conducted (RP5 VS EV5, RP6 VS EV6, RP7 VS EV7). For each comparison between the groups, the number of increase and decrease calls of each probe set was calculated using MS Excel and probe sets with the highest number of consistent changes among all samples were identified. Using these criteria, 115 genes showed statistically significant alterations in expression in at least two replicate studies. Gene lists were uploaded to GOTM, and functional annotation was performed. Further information on genes were obtained from public databases, such as NCBI. Hierarchical clustering was done using the clustering function (condition tree) in GeneSpring7.2 (Silicon Genetics, Inc., Redwood City, CA).
Primer sequences used in rtRT-PCR
5' CGGGAGCCGACTATGACTACTC 3'
5' GGGCTTAACTACCTGTTCAACTCTG 3'
5' ATTTCTTCTCGTTTTCACAGGC 3'
5' TCGGTCTTGTTCTCCCTCAG 3'
5' AACCCTGAAGCCATCACTG 3'
5' GCTTCGGACAAGTCTGTTATAG 3'
5' ACGCGTTGTGATCTCCTTCT 3'
5' GCACTATGATCCACTCCACC 3'
5' TGGCTTCTCACTTCAATCT 3'
5' CCACGGGTCCTGACTTTT 3'
5' AGCCCTTGGAGCCTCTGCA 3'
5' CGGAGCCAAAGATGGAGATCA 3'
5' TGATCCTCCAAGAATACG 3'
5' ACAAGTGTCGCAACAGAA 3'
5' GAAGACGGGAAGAAAGGG 3'
5' GTCGAGGTCACCGAAAGC 3'
5' CAATGAGACGGAGTTGACAG 3'
5' ACGCTGGTTCTTGATGTT 3'
5' TTGCAGATGCCGAGCAGCGT 3'
5' TGGGCTGAGGGCTAGGGCTG 3'
5' ATTGTCGGCTACTGCG 3'
5' ATTGTATGTTGGCTCC 3'
5' ACGGATTTGGTCGTATTGGG 3'
5' CGCTCCTGGAAGATGGTGAT 3
Cell differentiation induction assays
For sodium butyrate treatments, Caco2 cells were seeded into 60-mm culture dishes at 3 × 105 cells/plate. After 24 hr, cells were cultured in medium contained 4 mmol/L sodium butyrate. The cells were harvested at the time points of 24 hr, 48 hr, 72 hr after sodium butyrate stimulation. Expression of differentiation markers and IGFBP-rP1 were performed in the cell lysates and supernatants.
The ELISA method was carried out as described . Briefly, wells of microtiter plates were coated (for 18 hr at 4°C) with 100 ng/ml IGFBP-rP1 polyclonal antibody in 100 μl of coating buffer (0.05 M Na2CO3 and 0.05 M NaHCO3, pH 9.6) and were then blocked with 2% BSA in PBS for 1 hr at 37°C. Samples were diluted with 0.5% BSA (1:1) and a total of 100 μl was loaded in duplicates and incubated for 2 hr at room temperature, followed by the addition of 100 μl IGFBP-rP1 monoclonal antibody (200 ng/ml) for an additional 2 hr at room temperature. HRP-conjugated goat anti- mouse IgG (1:20,000) in blocking buffer was added (for 1 hr at room temperature) and the reaction was visualized by the addition of 100 μl of the chromogenic substrate (3,30,5,50- tetramethylbenzidine) for 30 min. The reaction was stopped with 100 μl H2SO4 and absorbance at 450 nm was measured with a reduction at 630 nm using ELISA plate reader. Plates were washed five times with washing buffer (PBS, pH 7.4, containing 0.1% (v/v) Tween 20) after each step. As a reference for quantification, a standard curve was established by a serial dilution of recombinant IGFBP-rP1 protein, ranging from 150 ng/ml to 1 ng/ml.
Statistical package SPSS (version 11.0) was applied. The Chi-square test was used to analyze the ranked data in tissue samples. Student't test was used in the cell line experiments. A value of P < 0.05 was considered statistically significant.
Expression pattern of IGFBP-rP1 in normal colonic epithelium and colorectal carcinoma
Comparison of IGFBP-rP1's expression in paired cancerous and normal tissues
IGFBP-rP1 expression in Cancerous tissue
IGFBP-rP1 expression in paired Normal tissue
Correlation between IGFBP-rP1 expression and differentiation in colorectal carcinoma
Morphology change of colorectal cancer cells induced by IGFBP-rP1
Regulation of well known differentiation markers by IGFBP-rP1
Gene expression profiles
Seventy-eight reproducible differentially expressed genes induced by IGFBP-rP1 in RKO cells
the cytoskeleton and actin binding
[NM_004342] CALD1 (1, 1.4, 0.7) *
[NM_024496] C14orf4 (-0.3,0.1(NC),-0.3)
[NM_001457] FLNB (0.4,1.4,-0.3)
[NM_032883] C20orf100 (-0.8,-0.2(NC),-0.6)
[NM_018027] FRMD4A (0.5,0.6,0.5) *
[NM_001964] EGR1 (0.7,0.4,-0.1)
[NM_005556] KRT7 (-0.2,-0.3,-0.2 (NC**))
[NM_002273] KRT8(-0.7,-0.3 (NC),-1)
[NM_005252] FOS (1.1,0.6,-0.5)
[NM_002281] KRTHB1 (-3.8,0.7,-1.3)
[NM_017445] H2BFS (0.5,0.7,0)
[NM_002628] PFN2 (0.3,-0.7,-0.8)
[NM_080593] HIST1H2BK (0.5,0.6,-0.1(NC))
[NM_006950] SYN1 (-1.7,-1.5,-2.7) *
[NM_002165] ID1 (-1.2,-1.2,-1) *
[NM_001001522] TAGLN (1,1.6,0.6) *
[NM_002167] ID3 (-0.3(NC),-0.9,-0.7)
[NM_014903] NAV3 (0.9,0.4,0.6) *
[NM_001657] AREG (-2.7,-1.6,-0.4) *
[NM_002135] NR4A1 (0.6,0.9,-0.6 (NC))
[NM_004406] DMBT1 (0.5,-1.4,-1)
[NM_006186] NR4A2 (0.4,1,-0.4)
[NM_006851] GLIPR1 (0.3,0.1,0)
[NM_003489] NRIP1 (-0.9,0.4, -0.3)
[NM_001553] IGFBP-rP1 (6.6,5.6,5.9) *
[NM_005902] SMAD3 (-0.5,0.1,-0.6)
[NM_003155] STC1 (-2.7,-3.8,-1.5) *
[NM_001005176] SP140 (1.1,1.3,0.6) *
[NM_003236] TGFA (-0.3, -0.5, -0.5)
[NM_006022] TSC22D1 (-0.3(NC), -0.6,-0.5)
[NM_003254] TIMP1 (-0.1(NC),-0.3, -0.6)
[NM_003255] TIMP2 (-4.5,-0.3 (NC),-1)
[NM_022164] TINAGL1 (-0.9,0.9,-0.4)
[NM_032744] C6orf105 (-5 (NC),-1.5,-2.3)
Cell proliferation and differentiation
[NM_000700] ANNEXIN A1 (-1.8,-1.2.-0.5(NC))
[NM_005127] CLEC2B (0.9,1.3,0.4(NC))
[NM_004936] CDKN2B (-0.8,-1.7, -0.8) *
[NM_001004023] DYRK3 (0.8,0.9,-0.3(NC))
[NM_018482] DDEF1 (0.3,0.6,0.1(NC))
[NM_001008493] ENAH (0.4,0.4,0.1(NC))
[NM_015675] GADD45B (1.1,0.8,0.2 (NC))
[NM_001005915] ERBB3 (-0.8,0.2(NC),-0.4)
[NM_003641] IFITM1(-0.7,-1.5,-0.4 (NC))
[NM_005330] HBE1 (4.3,-4.6,-1.5)
[NM_001025242] IRAK1 (-0.6 (NC), -1.5,-0.6)
[NM_005525] HSD11B1 (-3.1,-1.1,1.7)
[NM_014330] PPP1R15A (0.7,0.5, -0.2)
[NM_203434] IER5L (-0.7,-0.7,-0.7) *
[NM_173354] SNF1LK (-0.5,-0.2,0.5)
[NM_005544] IRS1 (-1.4,-0.7,-0.9) *
[NM_000346] SOX9 (-1.9,-1.5,-1.1) *
[NM_002288] LAIR2 (-0.9,-1.3,0.4)
[NM_002354] TACSTD1 (-0.7,-1.4,-1.9) *
[NM_002291] LAMB1 (-1.1,-0.6,-1) *
[NM_006669] LILRB1(-1.5,-1.2,-0.1 (NC))
[NM_004753] DHRS3 (-2.4,0.1 (NC),-1.6)
[NM_021070] LTBP3 (-1.1,0.5(NC),-0.6)
[NM_001005336] DNM1 (-0.6,-0.4,-0.3(NC))
[NM_138794] LYPLAL1 (0.4,0.4,-0.3(NC))
[NM_005261] GEM (0.8,0.5,0.1 (NC))
[NM_024979] MCF2L (-0.1(NC),-2.6,-3.8)
[NM_015590] GPATCH4 (0.3,0.4,-0.1(NC))
[NM_006818] MLLT11 (1,-1.4,-1)
[NM_003979] GPRC5A (-1.2,-0.1,-0.7)
[NM_176870] MT1K (-0.5,-0.2(NC),-0.6)
[NM_004637] RAB7 (0.5,0.5,-0.2)
[NM_000271] NPC1 (0.6,0.8,0.1)
[NM_002890] RASA1 (0.3,-0.4,-0.5)
[NM_002599] PDE2A (-2(NC),-1.7,-2.4)
[NM_007211] RASSF8 (0.4(MI***),1.9,2.5)
[NM_018444] PPM2C (-0.6,-0.5,0.2(NC))
[NM_005168] RND3 (1,0.7, 0.1(NC))
[NM_004155] SERPINB9 (-0.6,0.3 (NC),-0.9)
[NM_005415] SLC20A1 (0.2(NC), -0.3,-0.5)
Validation of a Subset of the differentially expressed genes by RT-PCR
Gene expression changes during differentiation induced by butyrate in Caco2 cells
The spatial organization of the colonic mucosa implies that in vivo the cells normally undergo a sequence of events which includes proliferation, differentiation, apoptosis, and extrusion. The continuously regenerating colonic epithelium is characterized by the proliferation of undifferentiated multipotent stem cells located at the base of the crypts and by shedding of mature fully differentiated cells at the luminal surface. It is during this translocation that differentiation occurs. Thus, to identify the differentiation associated function of IGFBP-rP1 in vivo, it was of interest to determine the localization of IGFBP-rP1-expressing cells in normal colonic epithelium. We here first reported that IGFBP-rP1 expresses specifically in the well-differentiated cells on the surface throughout the length of the intestine, strikingly matches the expression pattern of the differentiation marker of colon epithelium. Interestingly, IGFBP-rP1 expression is also well maintained and actually increased in colorectal cancer, when compared with the paired normal tissues, consistent with our previous study which performed in another collection of colon cancer samples (n = 78) . The discrepancy of up regulation of IGFBP-rP1 in colon cancer tissue and low expression in cell lines may be due to the regulation of IGFBP-rP1 by the microenvironment, which is now under investigation in our laboratory. The upregulation of IGFBP-rP1 in colon cancer tissue seems paradoxical to the well defined expression of IGFBP-rP1 in terminally differentiated cells at the normal intestinal surface. Colorectal carcinoma consists of different histology types with differentiation status, including tubular adenocarcinoma, papillary carcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, and undifferentiated carcinoma. The differentiation status of cancer can provide insights into the degree of malignancy, prognosis. We found that IGFBP-rP1 was strongly expressed in low grade colorectal carcinoma and weakly expressed in high grade colorectal carcinoma, indicating that the molecule is a potential differentiation-associated marker in colon cancer.
Differentiation characteristics of the colon epithelium include the emergence of a polarized morphology and the appearance of or a significant increase in brush border hydrola enzyme activities, such as AKP [36, 37]. In this study, we choose the three colon cancer cell lines without endogenous IGFBP-rP1 expression, RKO, SW620 and CW2 cell lines, to observe the transfection of IGFBP-rP1 cDNA on the differentiation status of the cells. RKO and CW2 cell lines are both derived from primary colon adenocarcinoma. SW620 cell line was initiated from a lymph node metastasis from the primary colon adenocarcinoma. We found that transfection of IGFBP-rP1 cDNA could induce the cells to a more pronounced anterior-posterior polarity morphology, accompanied by the increase of AKP activity both in the cell lysates and cell supernatants.
Interestingly, analysis of other well known colon epithelium differentiation markers showed cell type specificity. CEA, a tumor-associated antigen, is widely used serum biomarker for colorectal cancer. Interestingly, it has also been shown to correlate with the differentiation state of colon. In the normal colonic mucosa, CEA expression showed crypt-surface distribution. CEA expression was strong in surface epithelial cells and goblet cells of the upper crypts, while very weak in the mid crypt and at the base. Cell lines with high expression of CEA showed shuttle-shape morphologic changes with long or dendritic-like cytoplasmic processes, decreased cell growth and de novo tumor formation in nude mice xenograft. In colon cancer cell lines, it is also widely used as differentiation marker[39–43]. In our study, upregulated expression of CEA were induced by IGFBP-rP1 in SW620 and CW2 cells. No detectable expression of CEA induced by IGFBP-rP1 in RKO cells was observed. These findings suggested that the function of IGFBP-rP1 in regulating cell differentiation may be cell lineage specific. RKO cell line is a poorly differentiated colon carcinoma cell line developed by Michael Brattain. The AKP activity in RKO cells was lower than that of SW620 and CW2 cells. Although the AKP activity of PcDNA3.1(IGFBP-rP1)-RKO transfectants was higher than that of RKO cells, it was lower than that of PcDNA3.1(IGFBP-rP1)-SW620 and PcDNA3.1(IGFBP-rP1)-CW2 cells. Thus, the differentiation status may lower in RKO cells than in SW620 and CW2 cells. This may be could explain, at least in part, why the other well known differentiation marker such as CEA, could not be detected after IGFBP-rP1 transfection in RKO cells.
Interestingly, our findings were in consistent with the studies reported by Sprenger et al., which demonstrated the elongated appearance of high IGFBP-rP1 expressing clones in prostate cancer cells . The authors explained that the morphological change was correlated with an increased sensitivity to undergo apoptosis. However, based on our findings which showed the cell morphological change was associated with regulations of several epithelium differentiation associated markers, we thought that the morphological change was tightly associated with the differentiation induction process.
IGFBP-rP1 is a protein with secretary character. We observed that recombinant IGFBP-rP1 stimulation could mimic the effects of the transfection of IGFBP-rP1 cDNA, although the extent may not be as high. These findings indicated that the autocrine stimulation was part of the mechanism for the secretary protein. Other mechanisms may also be responsible for the biological behaviour of IGFBP-rP1.
Normal cells differentiate to gain in the properties required for organ or tissue functions. However, the differentiation program can be distorted. Malignant cells have a differentiation block that results in an accumulation of inappropriate or abnormal cell type (i.e., anaplasia). However, this process could partly be reversed. Colon cancer cells undergo terminal differentiation in response to diverse differentiation stimuli. It has been well demonstrated that sodium butyrate, the natural product of intestinal flora, is an typical inducer of colon cancer cell line to terminally differentiated cells (reviewed in ). Exposure to sodium butyrate induces morphological and biochemical changes consistent with a more differentiated state. One of these cell lines, namely Caco2, has been extensive used and characterized for its ability to differentiate under sodium butyrate stimulation. In our study, the upregulation of AKP activity and CEA expression confirmed the differentiation process, although the elongated polarity morphology change was not observed during the process. Different from the above RKO, SW620, and CW2 cells, Caco2 cells express endogenous IGFBP-rP1. Interestingly, upregulated expression of IGFBP-rP1 was detected during the differentiation process, which showed a good correlation with the most widely used differentiation marker AKP activity, again indicating IGFBP-rP1 a molecule associated with colon cancer differentiation
CDX2 is a key molecule for directing intestinal development and differentiation. A gradient of CDX2 expression formed in the crypt-villus axis, primarily in the villus. Overexpression of CDX2 leads to growth arrest accompanied by upregulation of several markers associated with intestinal differentiation[47, 48]. Decreased or absent expression of CDX2 were found in poorly differentiated colon carcinomas[49–51]. In colon cancer cell lines, it is also used as differentiation marker. We analyzed the expression of CDX2 during the differentiation process. Interestingly, downregulation of CDX2 induced by butyrate treatment in Caco2 cells was found. The RKO cells showed a slight but reproducible decrease (when performed six times) in levels of CDX2. While the SW620 and CW2 cells showed no detectable regulation on the CDX2 expression after transfection of IGFBP-rP1 cDNA, suggesting that restoration of CDX2 is not required for differentiation in these cell lines. Our findings were consistent with the studies of Oualtrough et al . In their study, no significant regulation of CDX2 was observed with the differentiation process in colon cell lines. No detectable gradient CDX2 expression along the axis of the crypt was found. These observations indicated that the differentiation associated function of CDX2 may act depend upon the cell type and may confer tissue specificity.
The studies presented here in vitro and in vivo demonstrated that IGFBP-rP1 was a potential molecule associated with colon epithelium cells differentiation, expanded the previous findings on the molecule's proliferation inhibition, apoptosis and senescence induction role. Based on different assumptions, several opposite models linking proliferation, cell death, and differentiation are currently coexisting . Cellular proliferation, differentiation, apoptosis and senescence are physiological processes that show overlapping properties [55–57]. Furthermore, several lines of evidence suggested that they are alternative, independent phenomena [58, 59]. The balance among proliferation, differentiation, senescence and apoptosis tightly regulated to maintain homeostasis of colon epithelium. Our findings extend our knowledge on IGFBP-rP1's role in this balance.
In this context, it is important to precisely identify the molecular signature for IGFBP-rP1 in colon cancer. Among the 78 reproducible differentially expressed genes identified, there were several genes whose altered expression induced by IGFBP-rP1 has been previously reported in prostate cancer cells by sprenger et al , such as IL8, KRT8. However, interestingly, SOX9 was found to be upregulated in IGFBP-rP1-transfected prostate cancer cells, while downregulated in IGFBP-rP1 transfected colon cancer cells, indicating the cell lineage specific regulation. DAG of the enriched GO categories demonstrated that the enzyme regulator activity genes together with cytoskeleton and actin binding genes were of great significance. The enzyme activity participated in many biomedical processes. The relation between the cytoskeleton and the differentiation process has been well demonstrated in different organ systems. Our findings provide a clue for IGFBP-rP1's possible function in these important biomedical processes. The upreguation of TAGLN and downregulation of SOX9, IRS1, P15, AREG, IER5L, KRT8 in RKO, SW620 and CW2 colon cancer cells indicated these genes may be the target molecules for the biological behaviour of IGFBP-rP1 in colon cancer. The sodium butyrate induced Caco2 differentiation process was accompanied by downregulation of IRS1 and SOX9. While other IGFBP-rP1 responsible genes exhibited different expression patterns via induction by sodium butyrate. It has been demonstrated that sodium butyrate induce cellular growth arrest, differentiation and apoptosis in colon cancer cells through various molecular mechanisms, including histone hyperacetylating [61, 62], nuclear factor kappaB (NF-kB) activation together with a defective beta1 integrin- focal adhesion kinase (FAK)- phosphatidylinositol 3'-kinase (PI3K) pathways signaling, downregulating extracellular signal-regulated kinase (ERK) phosphorylation  and induction of cyclin D3 and p21 expression. Our findings indicated that IGFBP-rP1 possiblely worked in different signaling pathways during the differentiation process. Our observations are consistent with the studies by Velcich et al. demonstrating that different inducers (12-O-tetradecanoylphorbol-13-acetate, forskolin, and sodium butyrate) modulate specific sets of markers in the process of differentiation induction, suggesting various inducers seem to utilize different intracellular pathways for induction of differentiation .
Downregulation of IRS1 and SOX9 by both IGFBP-rP1 and sodium butyrate in colon cancer cells indicated that these two genes may play important roles in the differentiation and apoptosis induction process in colon epithelium. IRS1 was a docking protein for both type 1 insulin-like growth factor receptor (IGF-IR) and insulin receptor. It is a key mediator of the actions of insulin and IGFs, sending a mitogenic, anti-apoptotic, and anti-differentiation signal [66, 67]. IRS-1 null mice exhibit reduced body growth and reduced growth of several organs including the intestine . Up regulation of IRS1 were found in colon cancer, while downregulation of IRS1 levels were found in differentiating cells [69, 70]. As to SOX9, previous studies showed the role of SOX9 in committed differentiation, such as chondrocyte differentiation, outer root sheath differentiation, and the formation of the hair stem cell compartment. Blache and colleagues reported that SOX9 can inhibit intestinal crypt differentiation in the colon . Contrary to the expression pattern of IGFBP-rP1 in the healthy human colon epithelium, our research group found that the expression of SOX9 is restricted to the proliferative, lower half of the crypt , consistent with the studies by Bastide et al.. Additional experiments demonstrating the exact roles of these two genes in mediating IGFBP-rP1's effect on differentiation should be performed.
Analysis of differentiation by tumor cells often provides valuable information for both the diagnosis and therapy of human cancers. One can envision very exciting times in the future as seeks to testing the role of IGFBP-rP1 using the knockout mice model. Because our current results are limited to colorectal tumorigenesis models, other studies will be needed to determine whether our findings apply to other organ systems. From a clinical point of view, specifically targeting and manipulating the function of IGFBP-rP1 may offer a novel approach to the differentiation therapy of colon cancer. Further experiments are under way to study the molecular mechanism underlying the observations reported here.
Conflict of interests
The authors declare that they have no competing interests.
This work was supported by grants from China National "973" program (2007CB914304), the National Scientific Foundation of China (30570840, 30770989, 30900236), and the Zhejiang Provincial Natural Science Foundation of China (D2080011, Y2090152). We thank Dr. Hancheng Zheng for his technical help in the microarray data analysis.
- Jones JI, Clemmons DR: Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev. 1995, 16: 3-34.PubMedGoogle Scholar
- Hwa V, Oh Y, Rosenfeld RG: The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr Rev. 1999, 20: 761-787. 10.1210/er.20.6.761PubMedGoogle Scholar
- Yamanaka Y, Wilson EM, Rosenfeld RG, Oh Y: Inhibition of insulin receptor activation by insulin-like growth factor binding proteins. J Biol Chem. 1997, 272: 30729-30734. 10.1074/jbc.272.49.30729View ArticlePubMedGoogle Scholar
- Oh Y, Nagalla SR, Yamanaka Y, Kim HS, Wilson E, Rosenfeld RG: Synthesis and characterization of insulin-like growth factor-binding protein (IGFBP)-7. Recombinant human mac25 protein specifically binds IGF-I and -II. J Biol Chem. 1996, 271: 30322-30325. 10.1074/jbc.271.48.30322View ArticlePubMedGoogle Scholar
- Murphy M, Pykett MJ, Harnish P, Zang KD, George DL: Identification and characterization of genes differentially expressed in meningiomas. Cell Growth Differ. 1993, 4: 715-722.PubMedGoogle Scholar
- Swisshelm K, Ryan K, Tsuchiya K, Sager R: Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid. Proc Natl Acad Sci USA. 1995, 92: 4472-4476. 10.1073/pnas.92.10.4472PubMed CentralView ArticlePubMedGoogle Scholar
- Akaogi K, Okabe Y, Sato J, Nagashima Y, Yasumitsu H, Sugahara K, Miyazaki K: Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells. Proc Natl Acad Sci USA. 1996, 93: 8384-8389. 10.1073/pnas.93.16.8384PubMed CentralView ArticlePubMedGoogle Scholar
- Yamauchi T, Umeda F, Masakado M, Isaji M, Mizushima S, Nawata H: Purification and molecular cloning of prostacyclin-stimulating factor from serum-free conditioned medium of human diploid fibroblast cells. Biochem J. 1994, 303 (Pt 2): 591-598.PubMed CentralView ArticlePubMedGoogle Scholar
- How HK, Yeoh A, Quah TC, Oh Y, Rosenfeld RG, Lee KO: Insulin-like growth factor binding proteins (IGFBPs) and IGFBP-related protein 1-levels in cerebrospinal fluid of children with acute lymphoblastic leukemia. J Clin Endocrinol Metab. 1999, 84: 1283-1287. 10.1210/jc.84.4.1283PubMedGoogle Scholar
- Vizioli MG, Sensi M, Miranda C, Cleris L, Formelli F, Anania MC, Pierotti MA, Greco A: IGFBP7: an oncosuppressor gene in thyroid carcinogenesis. Oncogene. 29: 3835-3844.Google Scholar
- Komatsu S, Okazaki Y, Tateno M, Kawai J, Konno H, Kusakabe M, Yoshiki A, Muramatsu M, Held WA, Hayashizaki Y: Methylation and downregulated expression of mac25/insulin-like growth factor binding protein-7 is associated with liver tumorigenesis in SV40T/t antigen transgenic mice, screened by restriction landmark genomic scanning for methylation (RLGS-M). Biochem Biophys Res Commun. 2000, 267: 109-117. 10.1006/bbrc.1999.1937View ArticlePubMedGoogle Scholar
- Watson MA, Gutmann DH, Peterson K, Chicoine MR, Kleinschmidt-DeMasters BK, Brown HG, Perry A: Molecular characterization of human meningiomas by gene expression profiling using high-density oligonucleotide microarrays. Am J Pathol. 2002, 161: 665-672.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Y, Pacyna-Gengelbach M, Ye F, Knosel T, Lund P, Deutschmann N, Schluns K, Kotb WF, Sers C, Yasumoto H: Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has potential tumour-suppressive activity in human lung cancer. J Pathol. 2007, 211: 431-438. 10.1002/path.2132View ArticlePubMedGoogle Scholar
- Sprenger CC, Damon SE, Hwa V, Rosenfeld RG, Plymate SR: Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor suppressor protein for prostate cancer. Cancer Res. 1999, 59: 2370-2375.PubMedGoogle Scholar
- Degeorges A, Wang F, Frierson HF, Seth A, Chung LW, Sikes RA: Human prostate cancer expresses the low affinity insulin-like growth factor binding protein IGFBP-rP1. Cancer Res. 1999, 59: 2787-2790.PubMedGoogle Scholar
- Seth A, Kitching R, Landberg G, Xu J, Zubovits J, Burger AM: Gene expression profiling of ductal carcinomas in situ and invasive breast tumors. Anticancer Res. 2003, 23: 2043-2051.PubMedGoogle Scholar
- Bieche I, Lerebours F, Tozlu S, Espie M, Marty M, Lidereau R: Molecular profiling of inflammatory breast cancer: identification of a poor-prognosis gene expression signature. Clin Cancer Res. 2004, 10: 6789-6795. 10.1158/1078-0432.CCR-04-0306View ArticlePubMedGoogle Scholar
- Luo MJ, Lai MD: Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH. World J Gastroenterol. 2001, 7: 726-731.PubMedGoogle Scholar
- Lin J, Lai M, Huang Q, Ma Y, Cui J, Ruan W: Methylation patterns of IGFBP7 in colon cancer cell lines are associated with levels of gene expression. J Pathol. 2007, 212: 83-90. 10.1002/path.2144View ArticlePubMedGoogle Scholar
- Ruan W, Xu E, Xu F, Ma Y, Deng H, Huang Q, Lv B, Hu H, Lin J, Cui J: IGFBP7 plays a potential tumor suppressor role in colorectal carcinogenesis. Cancer Biol Ther. 2007, 6: 354-359. 10.4161/cbt.6.3.3702View ArticlePubMedGoogle Scholar
- Ma Y, Lu B, Ruan W, Wang H, Lin J, Hu H, Deng H, Huang Q, Lai M: Tumor suppressor gene insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) induces senescence-like growth arrest in colorectal cancer cells. Exp Mol Pathol. 2008, 85: 141-145. 10.1016/j.yexmp.2008.04.005View ArticlePubMedGoogle Scholar
- Kato MV: A secreted tumor-suppressor, mac25, with activin-binding activity. Mol Med. 2000, 6: 126-135. 10.1007/s0089400060126PubMed CentralView ArticlePubMedGoogle Scholar
- Kato MV, Sato H, Tsukada T, Ikawa Y, Aizawa S, Nagayoshi M: A follistatin-like gene, mac25, may act as a growth suppressor of osteosarcoma cells. Oncogene. 1996, 12: 1361-1364.PubMedGoogle Scholar
- Sprenger CC, Vail ME, Evans K, Simurdak J, Plymate SR: Over-expression of insulin-like growth factor binding protein-related protein-1(IGFBP-rP1/mac25) in the M12 prostate cancer cell line alters tumor growth by a delay in G1 and cyclin A associated apoptosis. Oncogene. 2002, 21: 140-147. 10.1038/sj.onc.1205021View ArticlePubMedGoogle Scholar
- Wilson HM, Birnbaum RS, Poot M, Quinn LS, Swisshelm K: Insulin-like growth factor binding protein-related protein 1 inhibits proliferation of MCF-7 breast cancer cells via a senescence-like mechanism. Cell Growth Differ. 2002, 13: 205-213.PubMedGoogle Scholar
- Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR: Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell. 2008, 132: 363-374. 10.1016/j.cell.2007.12.032PubMed CentralView ArticlePubMedGoogle Scholar
- Tomlinson IP, Bodmer WF: Failure of programmed cell death and differentiation as causes of tumors: some simple mathematical models. Proc Natl Acad Sci USA. 1995, 92: 11130-11134. 10.1073/pnas.92.24.11130PubMed CentralView ArticlePubMedGoogle Scholar
- Corn PG, El-Deiry WS: Derangement of growth and differentiation control in oncogenesis. Bioessays. 2002, 24: 83-90. 10.1002/bies.10036View ArticlePubMedGoogle Scholar
- Vendrell JA, Magnino F, Danis E, Duchesne MJ, Pinloche S, Pons M, Birnbaum D, Nguyen C, Theillet C, Cohen PA: Estrogen regulation in human breast cancer cells of new downstream gene targets involved in estrogen metabolism, cell proliferation and cell transformation. J Mol Endocrinol. 2004, 32: 397-414. 10.1677/jme.0.0320397View ArticlePubMedGoogle Scholar
- Bertin R, Acquaviva C, Mirebeau D, Guidal-Giroux C, Vilmer E, Cave H: CDKN2A, CDKN2B, and MTAP gene dosage permits precise characterization of mono- and bi-allelic 9p21 deletions in childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2003, 37: 44-57. 10.1002/gcc.10188View ArticlePubMedGoogle Scholar
- Ernst T, Hergenhahn M, Kenzelmann M, Cohen CD, Bonrouhi M, Weninger A, Klaren R, Grone EF, Wiesel M, Gudemann C: Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. Am J Pathol. 2002, 160: 2169-2180.PubMed CentralView ArticlePubMedGoogle Scholar
- Plymate SR, Haugk KH, Sprenger CC, Nelson PS, Tennant MK, Zhang Y, Oberley LW, Zhong W, Drivdahl R, Oberley TD: Increased manganese superoxide dismutase (SOD-2) is part of the mechanism for prostate tumor suppression by Mac25/insulin-like growth factor binding-protein-related protein-1. Oncogene. 2003, 22: 1024-1034. 10.1038/sj.onc.1206210View ArticlePubMedGoogle Scholar
- Tohmiya Y, Koide Y, Fujimaki S, Harigae H, Funato T, Kaku M, Ishii T, Munakata Y, Kameoka J, Sasaki T: Stanniocalcin-1 as a novel marker to detect minimal residual disease of human leukemia. Tohoku J Exp Med. 2004, 204: 125-133. 10.1620/tjem.204.125View ArticlePubMedGoogle Scholar
- Shafat I, Zcharia E, Nisman B, Nadir Y, Nakhoul F, Vlodavsky I, Ilan N: An ELISA method for the detection and quantification of human heparanase. Biochem Biophys Res Commun. 2006, 341: 958-963. 10.1016/j.bbrc.2006.01.048PubMed CentralView ArticlePubMedGoogle Scholar
- Shao L, Huang Q, Luo M, Lai M: Detection of the differentially expressed gene IGF-binding protein-related protein-1 and analysis of its relationship to fasting glucose in Chinese colorectal cancer patients. Endocr Relat Cancer. 2004, 11: 141-148. 10.1677/erc.0.0110141View ArticlePubMedGoogle Scholar
- Boyd D, Florent G, Chakrabarty S, Brattain D, Brattain MG: Alterations of the biological characteristics of a colon carcinoma cell line by colon-derived substrata material. Cancer Res. 1988, 48: 2825-2831.PubMedGoogle Scholar
- Naishiro Y, Yamada T, Takaoka AS, Hayashi R, Hasegawa F, Imai K, Hirohashi S: Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of beta-catenin/T-cell factor 4-mediated gene transactivation. Cancer Res. 2001, 61: 2751-2758.PubMedGoogle Scholar
- Abbasi AM, Chester KA, MacPherson AJ, Boxer GM, Begent RH, Malcolm AD: Localization of CEA messenger RNA by in situ hybridization in normal colonic mucosa and colorectal adenocarcinomas. J Pathol. 1992, 168: 405-411. 10.1002/path.1711680411View ArticlePubMedGoogle Scholar
- Velcich A, Palumbo L, Jarry A, Laboisse C, Racevskis J, Augenlicht L: Patterns of expression of lineage-specific markers during the in vitro-induced differentiation of HT29 colon carcinoma cells. Cell Growth Differ. 1995, 6: 749-757.PubMedGoogle Scholar
- Guan RJ, Ford HL, Fu Y, Li Y, Shaw LM, Pardee AB: Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer. Cancer Res. 2000, 60: 749-755.PubMedGoogle Scholar
- Wang J, Yang Y, Xia HH, Gu Q, Lin MC, Jiang B, Peng Y, Li G, An X, Zhang Y: Suppression of FHL2 expression induces cell differentiation and inhibits gastric and colon carcinogenesis. Gastroenterology. 2007, 132: 1066-1076. 10.1053/j.gastro.2006.12.004View ArticlePubMedGoogle Scholar
- Yang L, Olsson B, Pfeifer D, Jonsson JI, Zhou ZG, Jiang X, Fredriksson BA, Zhang H, Sun XF: Knockdown of peroxisome proliferator-activated receptor-beta induces less differentiation and enhances cell-fibronectin adhesion of colon cancer cells. Oncogene. 29: 516-526.Google Scholar
- Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, Holden SA, Chen LB, Singer S, Fletcher C, Spiegelman BM: Differentiation and reversal of malignant changes in colon cancer through PPARgamma. Nat Med. 1998, 4: 1046-1052. 10.1038/2030View ArticlePubMedGoogle Scholar
- Augenlicht L, Velcich A, Heerdt BG: Short-chain fatty acids and molecular and cellular mechanisms of colonic cell differentiation and transformation. Adv Exp Med Biol. 1995, 375: 137-148.View ArticlePubMedGoogle Scholar
- Matsumoto H, Erickson RH, Gum JR, Yoshioka M, Gum E, Kim YS: Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2. Gastroenterology. 1990, 98: 1199-1207.PubMedGoogle Scholar
- Silberg DG, Swain GP, Suh ER, Traber PG: Cdx1 and cdx2 expression during intestinal development. Gastroenterology. 2000, 119: 961-971. 10.1053/gast.2000.18142View ArticlePubMedGoogle Scholar
- Suh E, Traber PG: An intestine-specific homeobox gene regulates proliferation and differentiation. Mol Cell Biol. 1996, 16: 619-625.PubMed CentralView ArticlePubMedGoogle Scholar
- Lorentz O, Duluc I, Arcangelis AD, Simon-Assmann P, Kedinger M, Freund JN: Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation. J Cell Biol. 1997, 139: 1553-1565. 10.1083/jcb.139.6.1553PubMed CentralView ArticlePubMedGoogle Scholar
- Hinoi T, Tani M, Lucas PC, Caca K, Dunn RL, Macri E, Loda M, Appelman HD, Cho KR, Fearon ER: Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon. Am J Pathol. 2001, 159: 2239-2248.PubMed CentralView ArticlePubMedGoogle Scholar
- Freund JN, Domon-Dell C, Kedinger M, Duluc I: The Cdx-1 and Cdx-2 homeobox genes in the intestine. Biochem Cell Biol. 1998, 76: 957-969. 10.1139/bcb-76-6-957View ArticlePubMedGoogle Scholar
- Baba Y, Nosho K, Shima K, Freed E, Irahara N, Philips J, Meyerhardt JA, Hornick JL, Shivdasani RA, Fuchs CS, Ogino S: Relationship of CDX2 loss with molecular features and prognosis in colorectal cancer. Clin Cancer Res. 2009, 15: 4665-4673. 10.1158/1078-0432.CCR-09-0401PubMed CentralView ArticlePubMedGoogle Scholar
- Domon-Dell C, Wang Q, Kim S, Kedinger M, Evers BM, Freund JN: Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells. Gut. 2002, 50: 525-529. 10.1136/gut.50.4.525PubMed CentralView ArticlePubMedGoogle Scholar
- Qualtrough D, Hinoi T, Fearon E, Paraskeva C: Expression of CDX2 in normal and neoplastic human colon tissue and during differentiation of an in vitro model system. Gut. 2002, 51: 184-190. 10.1136/gut.51.2.184PubMed CentralView ArticlePubMedGoogle Scholar
- Blagosklonny MV: Apoptosis, proliferation, differentiation: in search of the order. Semin Cancer Biol. 2003, 13: 97-105. 10.1016/S1044-579X(02)00127-XView ArticlePubMedGoogle Scholar
- Wier ML, Scott RE: Regulation of the terminal event in cellular differentiation: biological mechanisms of the loss of proliferative potential. J Cell Biol. 1986, 102: 1955-1964. 10.1083/jcb.102.5.1955View ArticlePubMedGoogle Scholar
- Abrams JM: Competition and compensation: coupled to death in development and cancer. Cell. 2002, 110: 403-406. 10.1016/S0092-8674(02)00904-2View ArticlePubMedGoogle Scholar
- Li ZR, Hromchak R, Mudipalli A, Bloch A: Tumor suppressor proteins as regulators of cell differentiation. Cancer Res. 1998, 58: 4282-4287.PubMedGoogle Scholar
- Gandarillas A: Epidermal differentiation, apoptosis, and senescence: common pathways?. Exp Gerontol. 2000, 35: 53-62. 10.1016/S0531-5565(99)00088-1View ArticlePubMedGoogle Scholar
- Mitra RS, Wrone-Smith T, Simonian P, Foreman KE, Nunez G, Nickoloff BJ: Apoptosis in keratinocytes is not dependent on induction of differentiation. Lab Invest. 1997, 76: 99-107.PubMedGoogle Scholar
- Bauer NG, Richter-Landsberg C, Ffrench-Constant C: Role of the oligodendroglial cytoskeleton in differentiation and myelination. Glia. 2009, 57: 1691-1705. 10.1002/glia.20885View ArticlePubMedGoogle Scholar
- Hinnebusch BF, Meng S, Wu JT, Archer SY, Hodin RA: The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. J Nutr. 2002, 132: 1012-1017.PubMedGoogle Scholar
- Richon VM, Emiliani S, Verdin E, Webb Y, Breslow R, Rifkind RA, Marks PA: A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proc Natl Acad Sci USA. 1998, 95: 3003-3007. 10.1073/pnas.95.6.3003PubMed CentralView ArticlePubMedGoogle Scholar
- Levy P, Robin H, Bertrand F, Kornprobst M, Capeau J: Butyrate-treated colonic Caco-2 cells exhibit defective integrin-mediated signaling together with increased apoptosis and differentiation. J Cell Physiol. 2003, 197: 336-347. 10.1002/jcp.10345View ArticlePubMedGoogle Scholar
- Davido DJ, Richter F, Boxberger F, Stahl A, Menzel T, Luhrs H, Loffler S, Dusel G, Rapp UR, Scheppach W: Butyrate and propionate downregulate ERK phosphorylation in HT-29 colon carcinoma cells prior to differentiation. Eur J Cancer Prev. 2001, 10: 313-321. 10.1097/00008469-200108000-00004View ArticlePubMedGoogle Scholar
- Siavoshian S, Segain JP, Kornprobst M, Bonnet C, Cherbut C, Galmiche JP, Blottiere HM: Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression. Gut. 2000, 46: 507-514. 10.1136/gut.46.4.507PubMed CentralView ArticlePubMedGoogle Scholar
- Baserga R: The contradictions of the insulin-like growth factor 1 receptor. Oncogene. 2000, 19: 5574-5581. 10.1038/sj.onc.1203854View ArticlePubMedGoogle Scholar
- White MF: The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem. 1998, 182: 3-11. 10.1023/A:1006806722619View ArticlePubMedGoogle Scholar
- Pete G, Fuller CR, Oldham JM, Smith DR, D'Ercole AJ, Kahn CR, Lund PK: Postnatal growth responses to insulin-like growth factor I in insulin receptor substrate-1-deficient mice. Endocrinology. 1999, 140: 5478-5487. 10.1210/en.140.12.5478PubMedGoogle Scholar
- Chang Q, Li Y, White MF, Fletcher JA, Xiao S: Constitutive activation of insulin receptor substrate 1 is a frequent event in human tumors: therapeutic implications. Cancer Res. 2002, 62: 6035-6038.PubMedGoogle Scholar
- Valentinis B, Romano G, Peruzzi F, Morrione A, Prisco M, Soddu S, Cristofanelli B, Sacchi A, Baserga R: Growth and differentiation signals by the insulin-like growth factor 1 receptor in hemopoietic cells are mediated through different pathways. J Biol Chem. 1999, 274: 12423-12430. 10.1074/jbc.274.18.12423View ArticlePubMedGoogle Scholar
- Blache P, van de Wetering M, Duluc I, Domon C, Berta P, Freund JN, Clevers H, Jay P: SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes. J Cell Biol. 2004, 166: 37-47. 10.1083/jcb.200311021PubMed CentralView ArticlePubMedGoogle Scholar
- Lu B, Fang Y, Xu J, Wang L, Xu F, Xu E, Huang Q, Lai M: Analysis of SOX9 expression in colorectal cancer. Am J Clin Pathol. 2008, 130: 897-904. 10.1309/AJCPW1W8GJBQGCNIView ArticlePubMedGoogle Scholar
- Bastide P, Darido C, Pannequin J, Kist R, Robine S, Marty-Double C, Bibeau F, Scherer G, Joubert D, Hollande F: Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium. J Cell Biol. 2007, 178: 635-648. 10.1083/jcb.200704152PubMed CentralView ArticlePubMedGoogle Scholar
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