Background
Methods
Generation of LP-1-derived clones
Cell cycle analysis by flow cytometry
Matrigel invasion assay
Clonogenicity assay
Immunoblotting
In vivo engraftment experiments
Immunohistochemistry of tumor sections
Microarray hybridization, gene expression data and statistical analyses
Real-time quantitative RT-PCR
Tumor engraftment onto chick chorio-allantoic membrane
Results
Cyclin D1b, cyclin K and c-Myc expressing LP-1-derived clones display tumorigenic properties
Cyclin D1b and cyclin K are not mitogenic in LP-1 cells
Cyclin D1b and cyclin K expression alter LP-1 cells transcriptome
Cyclin D1b and cyclin K alter cell cycle and survival genes expression
Gene | Microarray data | ΔCt (Ct LP-1D1b-Ct LP-1cl1) | Fold change | FC (microarray) |
---|---|---|---|---|
36B4
| nm* | -0.78/-0.52/-0.42** | 1 |
-
|
CSN2
| +7.33 | -2.16/-2.70 | +7.67/+6.34 |
+7.33
|
FGFR3
| +5.07 | -2.78/-1.62 | +11.79/+4.25 |
+5.09
|
FHIT
| +4.67 | -1.41/-1.10 | +3.68/+3.68 |
+4.67
|
HSP90B1
| +2.25 | -1.21 | +3.20 |
+2.26
|
TUBB2B
| -2.32 | 1.01 | -1.45 |
-2.32
|
TFRC
| -6.48 | 3.77/3.14 | -9.51/-6.36 |
-6.48
|
CD48
| -12.27 | 4.05/4.18 | -9.64/-13.08 |
-12.27
|
Gene
|
Microarray data
|
ΔCt (Ct LP-1K- Ct LP-1cl1)
|
Fold change
|
FC (microarray)
|
36B4
| nm* | -0.25/-0.40/-0.20/-0.36 | 1 |
-
|
LTB
| +40.66 | -5.7 | +43.71 |
+40.67
|
FN1
| +13.54 | -3.55 | +8.87 |
+13.54
|
BCL2
| +3.17 | -2.36 | +3.89 |
+3.17
|
CDK6
| -4.14 | 1.83/1.95 | -4.08/-4.43 |
-4.14
|
GAPDH
| -5.15 | 1.09 | -2.44 |
-6.15
|
UCHL1
| -63.82 | 11.11/11.25 | -2538/-2797 |
-63.82
|
Gene | Protein | LP-1D1b vs. cl1 | LP-1K vs. cl1 |
---|---|---|---|
SESN2
| Sestrin 2 | +3.36* | nm** |
DDIT3
| DNA-damage-inducible transcript 3 | +3.35 | nm |
CCNB1IP1
| Cyclin B1 interacting protein 1 | nm | +10.36 |
RASSF5
| Ras association (RalGDS/AP-6) domain family member 5 | nm | +4.79 |
CDNK1A
| Cyclin-dependent kinase inhibitor 1A | nm | +4.30 |
CABLES1
| CDK5 and ABL enzyme substract 1 | nm | +3.30 |
MAD2L1
| MAD2 mitotic arrest deficient-like 1 | nm | -3.13 |
CCNB2
| Cyclin B2 | nm | -3.14 |
GAS2
| Growth-arrest specific 2 | nm | -3.22 |
MK167
| Antigen identified by monoclonal antibody Ki67 | nm | -3.37 |
PINX1
| PIN2-interacting protein 1 | nm | -3.57 |
CCNF
| Cyclin F | nm | -3.65 |
CKS2
| CDC28 protein kinase regulatory subunit 2 | nm | -3.68 |
CHMP1A
| Chromatin modifying protein 1A | nm | -3.87 |
CDK6
| Cyclin-dependent kinase 6 | nm | -4.14 |
CCNB1
| Cyclin B1 | nm | -5.23 |
CDKN3
| Cyclin-dependent kinase inhibitor 3 | nm | -6.53 |
CDKN2C
| Cyclin-dependent kinase inhibitor 2C (p18) | nm | -8.18 |
CDKN2B
| Cyclin-dependent kinase inhibitor 2B (p15) | -3.75 | -2.83 |
Gene | Protein | LP-1D1b vs. cl1 | LP-1K vs. cl1 |
---|---|---|---|
FGFR3
| Fibroblast growth factor receptor 3 | +5.08* | nm** |
AKT3
| v-akt oncogene homolog 3 | nm | -5.16 |
MET
| HGFR, Met proto-oncogene | nm | -4.33 |
ITPKA
| Inositol 1,4,5-triphosphate 3-kinase A | nm | -3.94 |
CD81
| CD81 molecule | nm | +3.03 |
PIK3CG
| Phosphoinositide 3-kinase gamma | nm | +3.20 |
DOK6
| Docking protein 6 | nm | +3.21 |
MAPK13
| MAP kinase 13 | nm | +3.25 |
ECOP
| EGFR-overexpressed protein | nm | +3.39 |
PRKD2
| Protein kinase D2 | nm | +3.94 |
DUSP6
| Dual specificity phosphatase 6 | nm | +8.37 |
SKAP1
| Src kinase associated phosphoprotein | nm | +9.28 |
SYK
| Spleen tyrosine kinase | nm | +9.87 |
MAPK12
| MAP kinase 12 | nm | +10.10 |
BLK
| B lymphoid tyrosine kinase | +2.39 | +12.25 |
Cyclin K inhibits migration of LP-1-derived clones and enhances its clonogenic capacities
Gene | Protein | LP-1D1b vs. cl1 | LP-1K vs. cl1 |
---|---|---|---|
CX3CR1
| Chemokine (C-X-C motif) receptor 1 | nm* | +63.24** |
CD99
| CD99 molecule | nm | +52.47 |
FXYD5
| FXY domain containing ion transport regulator 5 | nm | +43.74 |
CD9
| CD9 molecule | nm | +35.80 |
SPON1
| Spondin 1 | nm | +21.37 |
CD4
| CD4 molecule | nm | +18.57 |
CXCL12
| Chemokine (C-X-C motif) ligand 12 | nm | +17.26 |
ARGDIB
| RhoGDP dissociation inhibitor (GDI) beta | nm | +13.55 |
FN1
| Fibronectin 1 | nm | +13.54 |
SUT3
| SUT homolog 3 | nm | +13.44 |
ICAM3
| Intracellular adhesion molecule 3 | nm | +13.34 |
LAMB3
| Laminin beta 3 | nm | +12.74 |
PCDH1
| Protocadherin 1 | nm | +12.05 |
CNTNAP2
| Contactin associated protein-like 2 | nm | +11.79 |
MCAM
| Melanoma cell adhesion molecule | nm | +10.70 |
CCL2
| Chemokine (C-C motif) ligand 2 | nm | +10.09 |
SYK
| Spleen tyrosine kinase | nm | +9.87 |
ANXA9
| Annexin A9 | nm | +9.33 |
LAMA3
| Laminin alpha 3 | nm | +8.46 |
LPXN
| Leupaxin | nm | +7.86 |
CD93
| CD93 molecule | nm | +7.60 |
ERBB2IP
| Erbb2 interacting protein | nm | +6.12 |
NRCAM
| Neuronal cell adhesion molecule | nm | +6.09 |
ITGB2
| Integrin beta 2 | nm | +6.08 |
PCDH1
| Protocadherin 1 | nm | +5.77 |
CD97
| CD97 molecule | nm | +5.38 |
ADAM23
| ADAM metallopeptidase domain 23 | nm | +4.90 |
CTGF
| Connective tissue growth factor | nm | +4.60 |
CD36
| CD36 molecule | nm | +4.53 |
NLGN1
| Neuroligin | nm | +4.50 |
CD44
| CD 44 molecule | nm | +4.21 |
CNTNAP2
| Contactin associated protein-like 2 | nm | +4.03 |
CD33
| CD33 molecule | nm | +4.02 |
HNT
| Neurtrimin | nm | +3.97 |
SELPLG
| Selectin P ligand | nm | +3.92 |
PKD2
| Polycystic kidney disease | nm | +3.80 |
SIGLEC7
| Sialic acid binding Ig-like lectin 7 | nm | +3.56 |
L1CAM
| L1 cell adhesion molecule | nm | +3.21 |
COL18A1
| Collagen type XVIII alpha 1 | nm | +3.20 |
ADAM15
| ADAM metallopeptidase domain 15 | nm | +3.18 |
CDSN
| Corneodesmosin | nm | +3.17 |
SIGLEC9
| Sialic acid binding Ig-like lectin 9 | nm | +3.11 |
NEO1
| Neogenin homolog 1 | nm | -3.22 |
TROAP
| Trophilin-associated protein | nm | -3.60 |
ITGA6
| Integrin alpha 6 | nm | -3.86 |
ITGAE
| Integrin alpha E | nm | -3.96 |
DST
| Distonin | nm | -4.00 |
COL24A1
| Collagen type XXIV alpha 1 | nm | -4.07 |
JAM3
| Junctional adhesion molecule 3 | nm | -4.26 |
PKP2
| Plakophilin 2 | nm | -5.11 |
SPN
| Sialophorin (CD43) | nm | -5.55 |
JAM2
| Junctional adhesion molecule 2 | nm | -12.09 |
ITGB7
| Integrin beta7 | -4.26 | +4.65 |
Cyclin D1b allows neo-angiogenesis of engrafted tumors
Gene | Protein | LP-1D1b vs. cl1 | LP-1K vs. cl1 |
---|---|---|---|
FGFR3
| Fibroblast growth factor receptor 3 | +5.08* | nm** |
TNFRSF10B
| Tumor necrosis factor receptor superfamily member 10 B | +3.52 | nm |
GATA4
| GATA binding protein 4 | +2.15 | nm |
WARS
|
Tryptophanyl-tRNA synthase
|
+4.01
|
nm
|
IGFBP3
| Insulin-like growth factor binding protein 3 | nm | +26.42 |
EPAS1
| Endothelial PAS domain protein 1 | nm | +17.39 |
CXCL12
| CXC chemokine ligand 12 | nm | +17.26 |
SLIT3
| SLIT homolog 3 | nm | +13.44 |
CCL2
| Chemokine ligand 2 | nm | +10.09 |
CXCL16
| CXC chemokine ligand 16 | nm | +6.67 |
F11R
| F11 receptor | nm | +5.56 |
RUNX1
| Runt-related transcription factor 1 | nm | +5.13 |
EGFL7
| EGF-like domain multiple 7 | nm | +4.68 |
CTGF
| Connective tissue growth factor | nm | +4.60 |
TNFSF13
| Tumor necrosis factor superfamily member 13 | nm | +4.57 |
SERPINB6
| Serpin peptidase inhibitor clade B member 6 | nm | +4.53 |
PDGFB
| Platelet-derived growth factor beta | nm | +3.99 |
CXCL3
| CXC chemokine ligand 3 | nm | +3.76 |
SERPINE2
| Serpin peptidase inhibitor clade E member 2 | nm | +3.40 |
SERPINB1
| Serpin peptidase inhibitor clade B member 1 | nm | +3.36 |
IL8
| Interleukin 8 | nm | +3.30 |
MMP13
| Matrix metallopeptidase 13 | nm | +3.12 |
IFNGR1
| Interferon gamma receptor 1 | nm | +3.06 |
HAND2
| Heart and neural crest derivatives | nm | -3.04 |
NRP2
| Neuropilin | nm | -3.25 |
PTPRF
| Protein tyrosine phosphatase receptor type F | nm | -4.20 |
RUNX1T1
| Runt-related transcription factor 1 translocated to 1 | nm | -4.79 |
ADM
| Adrenomedullin | nm | -6.66 |
ID1
| Inhibitor of DNA binding 1 | nm | -8.32 |
LAMA3
|
Laminin alpha 3
|
nm
|
+8.45
|
IFI16
|
Interferon gamma-inducible protein 16
|
nm
|
+4.96
|
JAG2
|
Jagged 2
|
nm
|
+4.77
|
ZFP36
|
Zinc finger protein 36
|
nm
|
+3.60
|
ZFP36L2
|
Zinc finger protein 36 C3H type-like 2
|
nm
|
+3.60
|
BMPR1A
|
Bone morphogenetic protein receptor type 1A
|
nm
|
-3.65
|
PTPRM
| Protein tyrosine phosphatase receprot type M | +2.11 | +10.38 |
JDB2
| Jun dimerization protein 2 | +3.70 | +3.74 |
IGF2BP1
| Insulin-like growth factor 2 binding protein 1 | -2.52 | -5.70 |
COL9A1
|
Collagen type IX alpha 1
|
-3.41
|
-4.87
|
COL24A1
|
Collagen type XXIV alpha 1
|
-2.42
|
-4.07
|
DAPK1
|
Death-associated protein kinase 1
|
+2.48
|
-3.06
|
Discussion
Cyclin D1b and cyclin K alter LP-1 cells metabolism
Cyclin D1b and cyclin K modulate gene transcription and translation within LP-1 cells
Gene | Protein | LP-1D1b vs. cl1 | LP-1K vs. cl1 |
---|---|---|---|
DDIT3
| DNA-damage-inducible transcript 3 | +3.34* | nm** |
ATF3
| Activating transcription factor 3 | +2.27 | nm |
XBP1
| X-box binding protein 1 | +2.24 | nm |
IRF8
| Interferon regulatory factor 8 | +2.21 | nm |
RUNX1
| Runt-related transcription factor 1 | nm | +5.13 |
TCF4
| Transcription factor 4 | nm | +4.40 |
MITF
| Microphtalmia-associated transcription factor | nm | +3.50 |
NOTCH2
| Notch homolog 2 | nm | +3.36 |
STAT5A
| Signal transduction and transcription factor 5A | nm | +2.49 |
ETV6
| Ets variant 6 | nm | +2.32 |
MAF
| v-maf musculoaponeurotic fibrosarcomaa oncogene homolog | nm | +2.25 |
MYC
| v-myc myelocytomatosis viral oncogene homolog | nm | +2.08 |
ETS2
| v-ets erythroblastosis virus E26 oncogene homolog | nm | +2.06 |
IRF4
| Interferon regulated factor 4 | nm | -2.25 |
RBPJ
| Recombination signal binding protein for Ig kappa region | nm | -2.38 |
REL
| v-rel reticuloendotheliosis viral onvogene homolog | nm | -3.63 |
RUNX2
| Runt-related transcription factor related 2 | nm | -4.80 |
MYBL2
| v-myb myeloblastosis viral oncogene homolog-like 2 | nm | -5.18 |
ATF4
| Activating transcription factor 4 | +2.75 | +2.64 |