research article sema6d expression and patient survival in...

Research ArticleSEMA6D Expression and Patient Survival in BreastInvasive Carcinoma

Dongquan Chen12 Yufeng Li12 Lizhong Wang23 and Kai Jiao3

1Division of Preventive Medicine Department of Medicine University of Alabama at Birmingham Birmingham AL 35294 USA2Comprehensive Cancer Center Department of Medicine University of Alabama at Birmingham Birmingham AL 35294 USA3Research Division Department of Genetics University of Alabama at Birmingham Birmingham AL 35294 USA

Correspondence should be addressed to Dongquan Chen dongquanuabedu and Kai Jiao kjiaouabedu

Received 12 February 2015 Accepted 26 March 2015

Academic Editor Ian S Fentiman

Copyright copy 2015 Dongquan Chen et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Breast cancer (BC) is the secondmost common cancer diagnosed inAmericanwomen and is also the second leading cause of cancerdeath in women Research has focused heavily on BC metastasis Multiple signaling pathways have been implicated in regulatingBC metastasis Our knowledge of regulation of BC metastasis is however far from complete Identification of new factors duringmetastasis is an essential step towards future therapy Our labs have focused on Semaphorin 6D (SEMA6D) which was implicatedin immune responses heart development and neurogenesis It will be interesting to know SEMA6D-related genomic expressionprofile and its implications in clinical outcome In this study we examined the public datasets of breast invasive carcinoma fromTheCancer GenomeAtlas (TCGA)We analyzed the expression of SEMA6D along with its related genes their functions pathwaysand potential as copredictors for BC patientsrsquo survival We found 6-gene expression profile that can be used as such predictors Ourstudy provides evidences for the first time that breast invasive carcinoma may contain a subtype based on SEMA6D expressionThe expression of SEMA6D gene may play an important role in promoting patient survival especially among triple negative breastcancer patients

1 Introduction

Breast cancer (BC) is the second most common cancerdiagnosed inAmericanwomen and is also the second leadingcause of cancer death in women [1 2] It is estimated that inthe developed world one in eight women will develop breastcancer in her lifetime [3 4] BC lethality is mainly causedby metastasis which accounts for approximately 90 of BCdeaths [5ndash10] Metastatic BC can be treated sometimes formany years but cannot be cured BC primarily metastasizesto the bone lungs regional lymph nodes liver and brainwith the most common site being the bone Research hasfocused heavily on BC metastasis for many years Multiplesignaling pathways such as TGF120573 Wnt Notch and EGFhave been implicated in regulating metastasis of BC cells [5ndash10] However our knowledge of regulation of BC metastasisis far from complete Identification of new factors that playcritical roles in drivinginhibiting metastatic progression is

an essential step toward fully understanding BC metasta-sis and will also provide novel therapeutic targetsreagentsagainst BC Our labs have focused on Semaphorins (SEMAs)especially SEMA6D SEMAs were implicated in immuneresponses heart development and neurogenesis [11ndash13] andrecently in BC metastases [14ndash17]

Semaphorins were initially recognized as phylogeneti-cally conserved neuronal guidance cues and their criticalregulatory roles in BC metastasis have rapidly emerged inrecent years Based on their sequence similarity Semaphorinsare classified into eight classes classes 1-2 are found in inverte-brates classes 3ndash7 comprise the vertebrate Semaphorins andclass V is encoded by viruses Class 2 3 and V Semaphorinsare secreted while all other members are membrane teth-ered through a single transmembrane domain [17ndash25] Thesignature structure of Semaphorins is the sim500 amino acid(aa) Sema domain which is a variant of the 120573-propellerfold revealed from structural studies [22] All Semaphorins

Hindawi Publishing CorporationInternational Journal of Breast CancerVolume 2015 Article ID 539721 10 pageshttpdxdoiorg1011552015539721

2 International Journal of Breast Cancer

except class V contain a PSI (Plexin-semaphorin-integrin)domain immediately to the C-terminal side of the Semadomain Different classes of Semaphorins may also containadditional functional motifs

Published studies regarding functions of Semaphorins inBC have mainly focused on class 3 secreted Semaphorins andSEMA4D [14ndash17] Our recent bioinformatic analysis by usingpublic datasets reveals for the first time the potential role ofSEMA6D in regulating BC pathogenesis The extracellularregion of SEMA6D which contains the Sema and PSIdomains can be released from the cell surface to act asa secreted cytokine with unknown molecular mechanisms[26 27] Thus SEMA6D may act both locally through cell-cell contacts and more distantly through diffusion of itscleaved ectodomain The primary receptors of SEMA6D arePLXNA1 and PLXNA4 which belong to the Plexin receptorfamily The extracellular domains of Plexins interact withSemaphorins while the C-terminal tails of Plexins medi-ate intracellular signal transduction Unlike many signalingpathways (such as the TGF120573SMAD pathway) there isno ldquocanonicalrdquo intracellular transduction cascade mediatingactivities of Semaphorin-Plexin signaling Many intracellularsignaling molecules such as GTPase activating proteinsGTPGDP exchange factors and various tyrosine kinasescan be activated andor inactivated by Semaphorin-Plexinsignaling in a context-dependent manner [18ndash25]

In this study we examine the public datasets from TheCancer Genome Atlas (TCGA) National Cancer Institute(NCI) for expression of SEMA6D along with genes that inter-act with SEMA6D Other genes coregulated with SEMA6Dwere analyzed for their function pathway and potentialas copredictors for BC patientsrsquo survival We found 6-geneexpression profile that can be used as such predictorsWe alsofound that SEMA6D expression correlated with the cancerstatus of triple negative (TNBC) markers (ER PR and Her2genes) The study shows the role of SEMA6D as potentialsurvival predictor especially in TNBC patients

2 Methods

21 Datasets TheCancer GenomeAtlas (TCGA)Data Portalwas used to download breast invasive carcinoma (BRCA)samples (119899 = 1 100) The RNAseqV2 level 3 data whichincludes fragments per kilobase of exon per million frag-ments mapped- (FPKM-) normalized gene level data wereused before statistics In addition idf and sdrf files were alsodownloaded for sample mapping and annotation Clinicaloutcomes data were downloaded for correlation and survivalanalysis

22 Gene Expression Data Analysis and Annotation Gene-level normalized expression data were used in PartekGenomic Suite (PGS St Louis MO) for additional normal-ization statistics and annotation The analysis of variance(ANOVA) methods were used for group comparisons Falsediscovery rate (FDR) correction (Benjamini-Hochbergmeth-ods) was applied for multiple hypothesis testing purposeOther statistical tools such as SAS (Cary NC) and IngenuityPathway Analysis (IPA Redwood City CA) were used for

pathway analysis and building gene-gene interaction net-work Heatmap was generated by using hierarchical cluster-ing methods after z-normalization

23 Survival Analysis A total of 140 patients with clinicaloutcomes data available (survival status months of sur-vival demographics and ER PR and HER2 status etc)were included in the analysis Among significant genesafter SEMA6D-high versus SEMA6D-low expression com-parisons we selected top 20 genes with the highest or lowestexpression levels to correlate with clinical outcomes Loga-rithm 2 based transformation of each gene was performedprior to any analysis The correlation among these 20 geneswas evaluated using Pearson correlation coefficient and sum-mary statistics were presented including mean with standarddeviation median and range Associations between level ofgenes and overall survival (OS) were assessed with Kaplan-Meier (K-M) curves and log-rank tests Each gene wasdichotomized as above or belowmedian level of expression inthe survival analysis Significant association was determinedat 5 type I error level Multiple comparisons were notexplicitly controlled for due to the small sample size andexploratory nature of the analysis

3 Results and Discussion

Semaphorins including members in subclass 3 andSEMA4D have emerged as critical signaling moleculesin regulating BC pathology [14ndash17] The potential roles ofother members of the Semaphorin family in BC have notbeen well addressed in the literature Our ongoing studiessuggest that SEMA6D plays a critical role in mediatingBone Morphogenetic Protein (BMP) signaling to regulateepithelial-mesenchymal-transition (EMT) by endocardialcells in developing hearts (Kai Jiao et alrsquos manuscript inpreparation) EMT is an essential step for initiatingmetastasisin BC and other cancersWe thus decided to apply a bioinfor-matic approach to examine the potential role of SEMA6Din BC tumorigenesis and progression using publically avail-able datasets After testing a few smaller datasets form GeneOntology Omnibus (GEO) we chose to use a breast invasivecarcinoma (BRCA) dataset from TCGA as it represents amajor public data source with clinical outcomes informationsuch as overall survival after diagnosis We divided all sam-ples (119899 = 1 100) into three roughly equal sized groups basedon SEMA6D expression (high medium and low) The com-parison of SEMA6D-high versus SEMA6D-low expressiongroup will reveal genes that are coregulated with SEMA6D

Gene Expression Profile by Principle Components Analysis(PCA) and Hierarchical Clustering To examine overall geneexpression profile and sample similarities we perform thePCA analysis of all samples As showed in Figure 1 thePCA showed a clear separation among SEMA6D-high (H)SEMA6D-medium (M) and SEMA6D-low (L) groups Thisindicates different gene expression profiles among the threegroups

Based on the genes that are differentially expressed inSEMA6D-high versus SEMA6D-low expression groups we

International Journal of Breast Cancer 3

PC

2 5

28

PC 1 795

PC 3 509

PCA mapping (183)

HLM

PC CC 3 509PCC

Figure 1 Principle component analysis (PCA) of all samples

then performed a hierarchical clustering (Figure 2) usinglog2-transformednormalized signal intensitiesTheobservedseparation of samples in each group (SEMA6D-highSEMA6D-medium and SEMA6D-low) indicates differentexpression profiles among these groups

Consistent with the PCA analysis the high SEME6Dexpression samples showed a congregation in the lower partof the figure which indicates a clear separation of samplesbased on SEMA6D expression In other words BC samplesmay contain a subtype with high SEMA6D expression

We further examined SEMA6D levels by includingSEMA6D-medium versus SEMA6D-low expression groupcomparison As shown in the Venn diagram in Figure 3 58unique genes are significant between SEMA6D-medium andSEMA6D-low patients while 2357 genes are significant inSEMA6D-high versus SEMA6D-low expression comparisonThis suggests that higher level of SEMA6D expression maylead tomore significant changes as evidenced by an increasednumber of genes with significant changes This also suggeststhat the function of SEMA6D may be dependent on theexpression level or that SEMA6D-induced functional effectsare dose-dependent

Among significant genes of SEMA6D-high versusSEMA6D-low comparison Gene Ontology (GO) analysis forbiological processes revealed that multicellular organismaldevelopment and G-protein coupled receptor protein signalingpathway are among the top changed GO biological processes(Table 1) It has been shown that SEMA6D may play a rolenot only during heart development [26 27] but also duringdevelopment of retina [28] and axon in zebra fish [29] Ourresults support these findings and showed that SEMA6Dplayed a major role in organismal development in additionto the G-protein coupled receptor signaling (Table 1)

The semaphorins and their receptors the neuropilins andthe Plexins are constituents of a complex regulatory systemthat controls axonal guidance [30] It was suggested that

Table 1 Biological process SEMA6D high versus low comparison

Biological process Enrichmentscore

Enrichment119901 value

Multicellular organismaldevelopment 3434 122119864 minus 15

G-protein coupled receptorprotein signaling pathway 3113 302119864 minus 14

Cell adhesion 1993 221119864 minus 09

Nervous systemdevelopment 1992 223119864 minus 09

Mitotic cell cycle 1976 263119864 minus 09

Cell division 1862 822119864 minus 09

Mitosis 1854 890119864 minus 09

M phase of mitotic cellcycle 1829 114119864 minus 08

Ion transport 1724 327119864 minus 08

Response to drug 1608 104119864 minus 07

Table 2Molecular function SEMA6Dhigh versus low comparison

Molecular function Enrichmentscore


Receptor activity 2290 114119864 minus 10

Sequence-specific DNAbinding 2127 580119864 minus 10

Voltage-gated sodiumchannel activity 2010 187119864 minus 09

Signal transducer activity 1714 359119864 minus 08

Calcium ion binding 1656 643119864 minus 08

Heparin binding 1578 140119864 minus 07

Voltage-gated ion channelactivity 1469 416119864 minus 07

Receptor binding 1443 543119864 minus 07

G-protein coupled receptoractivity 1260 336119864 minus 06

Sequence-specific DNAbinding TF activity 1160 914119864 minus 06

SEMA6D may bind to different receptor components andthus exert distinct functions during cardiac morphogenesis[26] Our results suggested a broad function of SEMA6Dto initiate signaling events that link to G-protein coupledreceptor (GPCR) signaling (Table 1) and overall receptoractivities (Table 2) GPCRs represent a super family of cellsurface signaling proteins and play essential roles in cancermetastasis [2 31 32] and are one of themost promising targetsof metastatic breast cancer therapy [33ndash37]

In cell adhesion cell-cell interactions between cancer cellswith endothelium determine the metastatic spreadThere aretwomajor cell adhesions including selectin and integrin andaccumulating evidence confirms that tumor cell interactionsthrough them actively contribute to the metastatic spreadof tumor cells [38] Our results suggest a pivotal role forSEMA6D in tumor metastasis especially receptor activities(Table 2) and GPCR signaling (Table 1)


500000minus500

L

L

L

L

LLL

L

LL

L

L

L

L

L

L

L

L

L

L

L

H

H

H

H

M

M

MM

M

M

M

MMM

M

M

M

M

M

M

M

Hierarchical clustering

Figure 2 Hierarchical clustering of significant genes of SEMA6D-H versus -L expression Genes (vertical high expression in red and lowexpression in green) and samples (horizontal SEMA6D-high in green SEMA6D-medium in blue and SEMA6D-low in brown)were clusteredbased on Euclidean dissimilarity matrix

58 255 2357

M versus L FDR H versus L FDRp lt 005 FC gt 2 (n = 313) p lt 005 FC gt 2 (n = 2612)

Figure 3 Number of significant genes between the two compar-isons H versus L and M versus L FC fold change

TheGO-molecular functions also reveal that receptor activ-ity sequence-specific DNA binding and voltage-gated sodiumchannel activities are among top affected molecular functionswhen SEMA6D level is high (Table 2) These results suggestthat SEMA6D may initiate member receptors activation asa ligand The gene-gene interactions among those genesthat directly or indirectly interact with SEMA6D partiallyconfirmed this hypothesis As shown in Figure 4 elevatedPLXNA4 may lead to an increase of SEMA6D expressionand trigger transcriptions by the general transcription factorsFOS and FOXO1 This may lead to a cascade of activationsof membrane receptors including G-protein coupled receptors(Table 2)

As reported Plexin-B1 is a receptor for the transmem-brane semaphorin SEMA4D (CD100) [39] and PLXNA4negatively regulates T lymphocyte responses [40] It hasbeen shown that SEMA6D induces NF-120581B transcriptional


Plasma membrane

Cytoplasm

Nucleus

PLXNA1

PLXNA4

5428

16518

AKT2

AKT1

PI3K (complex)

AKT3

FOSL1

SRF FOS

FOXO1

2711

4908

Sos

ABL1

SEMA6D Enah

Figure 4 Activation of SEMA6D and transcription The gene-gene interaction network was built based on direct interactions by usingIngenuity Pathway Analysis (IPA) suite Color indicates increased (in red) expression when SEMA6D-high samples were compared withSEMA6D-low samples The number indicated the fold changes of this comparison

activity in nonmalignantmesothelial cells [30] Two potentialtargets of SEMA6D the general transcription factors FOSand FOXO1 were both increased in SEME6D-high patientsFOXO1 has been widely reported in tumor oncogenesisand metastasis [41ndash43] This suggests an important role forSEMA6D in promoting general transcription through FOScoupledwith FOXO1 as previously reported [44]The balanceof transcriptions of both tumor suppressors and oncogenesmay be the key to understand the underlining mechanism

SEMA6D and Tumor Metastasis SEMA6D plays an impor-tant role in tissue development and differentiation a processinvolving epithelial-mesenchymal-transition (EMT) it willbe interesting to know if EMT-related genes are coregulatedin SEMA6D-high patients As shown in Table 3 majortumor metastatic promoter- (MMP-) 9 was dramaticallyreduced among SEMA6D-high samples while several tumor

metastatic promoters such as TGF-120573-related factors ZEB2ZEB1 and GNG11 however were elevated corresponding toa high level of SEMA6D In addition the expressions of allthese genes (except for DSC2) are highly correlated with theexpression of SEMA6D (Table 4) As SEMA6D was impli-cated in VEGF-dependent and anchorage-independent cellgrowth [30] we also included VEGF genes in the correlationanalysis High levels of correlations between SEMA6D leveland VEGFs were found as well (Table 4) suggesting a role ofVEGF family genes in mediating SEMA6D signaling

MMP family proteins especially MMP9 were suggestedto be involved in the process of metastasis of breast cancerto the brain [45] CD147-mediated metastasis in MCF7 cells[19] TGF120573-mediated signaling at the tumor-bone interface[46] and L2-mediated matrix remodeling in metastasis andmammary gland involution [47] Decreased autocrine EGFRsignaling in metastatic breast cancer cells inhibits tumor


Table 3 Expression of top EMT-related genes in SEMA6D-H versus L comparison

Symbol Description 119901 value(H versus L)

Foldchange

Fold(description)

MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kDa gelatinase 92 kDatype IV collagenase) 0163673 minus361

H downversus L

TMEM132A Transmembrane protein 132A 587119864 minus 27 minus221H downversus L

BMP7 Bone Morphogenetic Protein 7 285119864 minus 05 minus179H downversus L

DSC2 Desmocollin 2 855119864 minus 07 minus174H downversus L

HPRT1 Hypoxanthine phosphoribosyltransferase 1 782119864 minus 46 minus172H downversus L

KRT19 Keratin 19 144119864 minus 15 minus164H downversus L

SPP1 Secreted phosphoprotein 1 0000141 minus155H downversus L

PPPDE2 PPPDE peptidase domain containing 2 602119864 minus 24 minus148H downversus L


CDH1 Cadherin 1 type 1 E-cadherin (epithelial) 938119864 minus 11 minus146H downversus L

COL3A1 Collagen type III alpha 1 253119864 minus 15 189 H up versus L

MMP2 Matrix metallopeptidase 2 (gelatinase A 72 kDa gelatinase 72 kDatype IV collagenase) 374119864 minus 25 197 H up versus L

SNAI2 Snail homolog 2 (Drosophila) 828119864 minus 24 208 H up versus LMITF Microphthalmia-associated transcription factor 133119864 minus 42 208 H up versus LTCF4 Transcription factor 4 217119864 minus 75 235 H up versus LAHNAK AHNAK nucleoprotein 598119864 minus 57 237 H up versus LZEB2 Zinc finger E-box binding homeobox 2 155119864 minus 44 251 H up versus LZEB1 Zinc finger E-box binding homeobox 1 214119864 minus 73 267 H up versus LGNG11 Guanine nucleotide binding protein (G protein) gamma 11 131119864 minus 54 330 H up versus L

growth in bone and mammary fat pad through MMP9-dependent pathways [48] By using an RNA interferenceapproach the reduced levels of MMP-9 mRNA and proteincorrelated with inhibited phenotype of tumor invasion andmetastasis [14] Our results are in line with these findings andsuggest a tumor suppressor function for SEMA6D

On the other hand our results also showed an increasedexpression among SEMA6D-high samples of some importanttumor promoters such as ZEB12 which had been reported topromote EMT by modulating Zeb12 and TGF120573 expression[49] Our results thus strongly suggest that the balancebetween tumor suppressors and promoters is the key tounderstand the role of SEMA6D during EMT Anotherexplanation is that the increased expression of SEMA6Dmaybe the results not the cause ZEB12 changes and vice versa

SEMA6D Expression and Affected Signaling PathwaysAlthough roles of SEMAs have been suggested in breast can-cer [14ndash17] prostate cancer [50] and malignant mesotheli-oma [30] the underlying functional mechanisms includingpathways are largely unknown Nevertheless SEMA6D has

been reported to play a role in immune responses [12] NF-120581Bsignaling [30] and stromal expression of SEMA6D [50] Ourresults implicated top canonical pathways (Table 5) whichpartially confirmed previous reports such as cAMP-mediatedsignaling in cervical cancer cell migration [51] and in lungcancer [52] G-protein coupled receptor signaling in breastcancer [34 53] and adhesion and diapedesis in a breastcancer cell line [54] Therefore SEMA6D may play multipleroles during these processes although additional studies maybe needed to further delineate SEMA6D functions in thesepathways

SEMA6D Expression Correlates with Patientsrsquo Survival Inorder to determine if SEMA6D and SEMA6D-related genesare correlated with overall patient survival we filtered thesignificant gene list after SEMA6D-high versus SEMA6D-low expression comparison by choosing the top 10 mostupregulated genes and top 10 most downregulated genes andconducted a survival analysis by using K-M methods Wefound that 6 candidate genes were significantly associatedwith overall survival These genes are SEMA6D CLEC9A


Table 4 Correlation of SEMA6D with EMT gene expressions

Symbol 119903 119901 valueGNG11 055 127119864 minus 88

ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79

HPRT1 minus049 112119864 minus 68

SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55

TMEM132A minus043 105119864 minus 49

VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30

PPPDE2 minus031 270119864 minus 25

MMP9 minus027 718119864 minus 20

KRT19 minus026 277119864 minus 18

SPP1 minus022 870119864 minus 14

COL3A1 017 102119864 minus 08

VEGFA minus015 276119864 minus 07

CDH1 minus015 108119864 minus 06

VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209

119903 Spearman correlation coefficient n = 1100

Table 5 Canonical signaling pathway by SEME6D high expression

Pathway name 119901 value RatiocAMP-mediated signaling 227119864 minus 09 53222G-Protein coupled receptor signaling 260119864 minus 07 54265Granulocyte adhesion and diapedesis 273119864 minus 07 41176Agranulocyte adhesion and diapedesis 152119864 minus 06 41187Gas signaling 183119864 minus 05 27119

Table 6 Correlation of gene expression with patientsrsquo survival

Variable 119873 Mean SD Median Min Max Log-rank 119901SEMA6D 140 715 190 695 211 1209 00156

CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162

CBX2 140 771 193 736 281 1160 00709OS overall survival stratified by high (gtmedium) or low (ltmedium)expression p lt 005

Product-limit survival estimates10

08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects

Event Censored Median survival (95 CI)

70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian

Figure 5 SEMA6D correlates with patient survival

C10orf107 DONSON CHAC1 and TUBA1C Two moregenes COL4A6 and CBX2 genes are in borderline to be sig-nificant A summary of these 8 genes is listed in Table 6 andFigure 5

In addition increased expressions of SEMA6D CLEC9ACOL4A6 and C10orf107 are associated with better sur-vival while decreased expressions of DONSON CHAC1TUBA1C and CBX2 also correlate to better survival Figure 5showed survival probability based on SEMA6D expression(gemedian or lemedian expressions) Similar significant sep-aration trends were also observed for both CLEC9A andC10orf107 as positive predictors and DONSON CHAC1 andTUBA1C as negative predictors (data not shown)

Correlation of Expressions of SEMA6D and Other Genes withTNBC Status As only 30 of women with metastases survivefive years and virtually all TNBCwomenwill ultimately die oftheir disease despite systemic therapy [55] we further explorethe role of SEMA6D in promoting survival in TNBC patientsWe found not only that these genes associated with survivalbut also that they are interacting with TNBC status (Yes orNo) significantly for SEMA6D for example with a log-rank119901 = 00083 as shown in Figure 6 It is clearly shown thatTNBC patients (Figure 6 SEMA6D-high in brown relativeto SEMA6D-low in green) show larger survival differencesas compared with non-TNBC patients (SEMA6D-high inred relative to SEMA6D-low in blue) Other genes such asCLEC9A (119901 = 00083) and C10orf107 (119901 = 00083) aresimilarly associated with TNBC status (data not shown)

These results strongly suggest that SEMA6D expressionlevels correlate with overall survival (Figure 5) especially inTNBC patients (Figure 6)



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity Log-rank p = 00083

OS_Yr

TRIPLE_NEGATIVE=NO MEDlog2SEMA6D=leMEDIANTRIPLE_NEGATIVE=NO MEDlog2SEMA6D=gtMEDIANTRIPLE_NEGATIVE=YES MEDlog2SEMA6D=leMEDIANTRIPLE_NEGATIVE=YES MEDlog2SEMA6D=gtMEDIAN

TRIPLE_NEGATIVE=NO MEDlog2SEMA6D=leMEDIANTRIPLE_NEGATIVE=NOMEDlog2SEMA6D=gtMEDIANTRIPLE_NEGATIVE=YES MEDlog2SEMA6D=leMEDIANTRIPLE_NEGATIVE=YES MEDlog2SEMA6D=gtMEDIAN

Numberof subjects Censored Median survival

(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6

Figure 6 Interaction of SEMA6D with TNBC in patientsrsquo survival

4 Conclusions

Our study provides evidences that breast invasive carcinoma(BRCA) may contain a subtype based on SEMA6D expres-sionThe expression of SEMA6D genemay play an importantrole in promoting patient survival especially among triplenegative breast cancer (TNBC) patients

Abbreviations

TNBC Triple negative breast cancerBRCA Breast invasive carcinomaBC Breast cancerSEMA6D Semaphorin 6DBMP Bone Morphogenetic ProteinEMT Epithelial-mesenchymal-transitionPCA Principle component analysisGO Gene OntologyTCGA The Cancer Genome Atlas

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Dongquan Chen and Yufeng Li contributed equally to thestudy

Acknowledgments

The study was partially supported by institutional funding byUniversity of Alabama at Birmingham (UAB) to DongquanChen and a Faculty Development Grant from UAB Compre-hensive Cancer Center to Kai Jiao an R01 (HL095783) to KaiJiao and an R21 (CA179282) to Lizhong Wang

References

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[2] C DeSantis J Ma L Bryan and A Jemal ldquoBreast cancer statis-tics 2013rdquo CA Cancer Journal for Clinicians vol 64 no 1 pp52ndash62 2014

[3] J Ferlay D M Parkin and E Steliarova-Foucher ldquoEstimates ofcancer incidence and mortality in Europe in 2008rdquo EuropeanJournal of Cancer vol 46 no 4 pp 765ndash781 2010

[4] F Lalloo and D G Evans ldquoFamilial breast cancerrdquo ClinicalGenetics vol 82 no 2 pp 105ndash114 2012

[5] M Bendre D Gaddy R W Nicholas and L J Suva ldquoBreastcancer metastasis to bone it is not all about PTHrPrdquo ClinicalOrthopaedics and Related Research no 415 pp S39ndashS45 2003

[6] A J Redig and S S McAllister ldquoBreast cancer as a systemicdisease a view of metastasisrdquo Journal of Internal Medicine vol274 pp 113ndash126 2013

[7] O J Scully B-H Bay G Yip and Y Yu ldquoBreast cancer meta-stasisrdquo Cancer Genomics amp Proteomics vol 9 no 5 pp 311ndash3202012

[8] S Byler S Goldgar S Heerboth et al ldquoGenetic and epigeneticaspects of breast cancer progression and therapyrdquo AnticancerResearch vol 34 no 3 pp 1071ndash1077 2014

[9] M A Blanco and Y Kang ldquoSignaling pathways in breast cancermetastasismdashnovel insights from functional genomicsrdquo BreastCancer Research vol 13 no 2 p 206 2011

[10] J Izrailit and M Reedijk ldquoDevelopmental pathways in breastcancer and breast tumor-initiating cells therapeutic implica-tionsrdquo Cancer Letters vol 317 no 2 pp 115ndash126 2012

[11] R J Pasterkamp ldquoGetting neural circuits into shape with sem-aphorinsrdquo Nature Reviews Neuroscience vol 13 no 9 pp 605ndash618 2012

[12] B P OrsquoConnor S-Y Eun Z Ye et al ldquoSemaphorin 6D regulatesthe late phase of CD4+ T cell primary immune responsesrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 105 no 35 pp 13015ndash13020 2008

[13] J R Leslie F Imai K Fukuhara et al ldquoEctopic myelinatingoligodendrocytes in the dorsal spinal cord as a consequenceof altered semaphorin 6D signaling inhibit synapse formationrdquoDevelopment vol 138 no 18 pp 4085ndash4095 2011

[14] L Tamagnone ldquoEmerging role of semaphorins as major regula-tory signals and potential therapeutic targets in cancerrdquo CancerCell vol 22 no 2 pp 145ndash152 2012

[15] M Rehman and L Tamagnone ldquoSemaphorins in cancer bio-logical mechanisms and therapeutic approachesrdquo Seminars inCell amp Developmental Biology vol 24 no 3 pp 179ndash189 2013

[16] C Gu and E Giraudo ldquoThe role of semaphorins and theirreceptors in vascular development and cancerrdquo ExperimentalCell Research vol 319 no 9 pp 1306ndash1316 2013


[17] G C Harburg and L Hinck ldquoNavigating breast cancer axonguidance molecules as breast cancer tumor suppressors andoncogenesrdquo Journal of Mammary Gland Biology and Neoplasiavol 16 no 3 pp 257ndash270 2011

[18] U Yazdani and J R Terman ldquoThe semaphorinsrdquo Genome Biol-ogy vol 7 no 3 article 211 2006

[19] T S Tran A L Kolodkin and R Bharadwaj ldquoSemaphorinregulation of cellular morphologyrdquo Annual Review of Cell andDevelopmental Biology vol 23 pp 263ndash292 2007

[20] A Casazza P Fazzari and L Tamagnone ldquoSemaphorin sig-nals in cell adhesion and cell migration functional role andmolecular mechanismsrdquo Advances in Experimental Medicineand Biology vol 600 pp 90ndash108 2007

[21] R P Kruger J Aurandt and K-L Guan ldquoSemaphorins com-mand cells tomoverdquoNature ReviewsMolecular Cell Biology vol6 no 10 pp 789ndash800 2005

[22] EGherardi CA Love RM Esnouf andE Y Jones ldquoThe semadomainrdquoCurrentOpinion in Structural Biology vol 14 no 6 pp669ndash678 2004

[23] L Tamagnone and P M Comoglio ldquoTo move or not to moveSemaphorin signalling in cell migrationrdquo EMBO Reports vol 5no 4 pp 356ndash361 2004

[24] A Ahmed and B J Eickholt ldquoIntracellular kinases in sema-phorin signalingrdquo Advances in Experimental Medicine andBiology vol 600 pp 24ndash37 2007

[25] T Toyofuku and H Kikutani ldquoSemaphorin signaling duringcardiac developmentrdquo Advances in Experimental Medicine andBiology vol 600 pp 109ndash117 2007

[26] T Toyofuku H Zhang A Kumanogoh et al ldquoDual roles ofSema6D in cardiac morphogenesis through region-specificassociation of its receptor Plexin-A1 with off-track and vascularendothelial growth factor receptor type 2rdquo Genes and Develop-ment vol 18 no 4 pp 435ndash447 2004

[27] T Toyofuku H Zhang A Kumanogoh et al ldquoGuidance ofmyocardial patterning in cardiac development by Sema6Dreverse signallingrdquo Nature Cell Biology vol 6 no 12 pp 1204ndash1211 2004

[28] R L Matsuoka L O Sun K-I Katayama Y Yoshida and AL Kolodkin ldquoSema6B Sema6C and Sema6D expression andfunction during mammalian retinal developmentrdquo PLoS ONEvol 8 no 4 Article ID e63207 2013

[29] M Kimura M Taniguchi Y Mikami et al ldquoIdentification andcharacterization of zebrafish semaphorin 6Drdquo Biochemical andBiophysical Research Communications vol 363 no 3 pp 762ndash768 2007

[30] A Catalano R Lazzarini S D Nuzzo S Orciari and AProcopio ldquoTheplexin-A1 receptor activates vascular endothelialgrowth factor-receptor 2 and nuclear factor-kappaB to medi-ate survival and anchorage-independent growth of malignantmesothelioma cellsrdquo Cancer Research vol 69 no 4 pp 1485ndash1493 2009

[31] S Majumder M P Sowden S A Gerber et al ldquoG-protein-coupled receptor-2-interacting protein-1 is required for endo-thelial cell directional migration and tumor angiogenesis viacortactin-dependent lamellipodia formationrdquo ArteriosclerosisThrombosis and Vascular Biology vol 34 no 2 pp 419ndash4262014

[32] H-Y Li X-Y CuiWWu et al ldquoPyk2 and Srcmediate signalingto CCL18-induced breast cancer metastasisrdquo Journal of CellularBiochemistry vol 115 no 3 pp 596ndash603 2014

[33] S-H Chan W-C Huang J-W Chang et al ldquoMicroRNA-149targets GIT1 to suppress integrin signaling and breast cancermetastasisrdquo Oncogene vol 33 pp 4496ndash4507 2014

[34] W Wei Z-J Chen K-S Zhang et al ldquoThe activation of Gprotein-coupled receptor 30 (GPR30) inhibits proliferation ofestrogen receptor-negative breast cancer cells in vitro and invivordquo Cell Death amp Disease vol 5 Article ID e1428 2014

[35] J-M Renoir V Marsaud and G Lazennec ldquoEstrogen receptorsignaling as a target for novel breast cancer therapeuticsrdquoBiochemical Pharmacology vol 85 no 4 pp 449ndash465 2013

[36] N Dey B R Smith and B Leyland-Jones ldquoTargeting basal-likebreast cancersrdquo Current Drug Targets vol 13 no 12 pp 1510ndash1524 2012

[37] J-M Renoir ldquoEstradiol receptors in breast cancer cells asso-ciated co-factors as targets for new therapeutic approachesrdquoSteroids vol 77 no 12 pp 1249ndash1261 2012

[38] H Laubli and L Borsig ldquoSelectins promote tumor metastasisrdquoSeminars in Cancer Biology vol 20 no 3 pp 169ndash177 2010

[39] L Tamagnone S Artigiani H Chen et al ldquoPlexins are a largefamily of receptors for transmembrane secreted and GPI-anchored semaphorins in vertebratesrdquo Cell vol 99 no 1 pp71ndash80 1999

[40] M Yamamoto K Suzuki T Okuno et al ldquoPlexin-A4 negativelyregulates T lymphocyte responsesrdquo International Immunologyvol 20 no 3 pp 413ndash420 2008

[41] J Yang T Li C Gao et al ldquoFOXO1 31015840UTR functions as a ceRNAin repressing the metastases of breast cancer cells via regulatingmiRNA activityrdquo FEBS Letters vol 588 no 17 pp 3218ndash32242014

[42] X Wang C Lin X Zhao et al ldquoAcylglycerol kinase promotescell proliferation and tumorigenicity in breast cancer via sup-pression of the FOXO1 transcription factorrdquoMolecular Cancervol 13 article 106 2014

[43] X Feng Z Wu Y Wu et al ldquoCdc25A regulates matrix metal-loprotease 1 through Foxo1 and mediates metastasis of breastcancer cellsrdquoMolecular and Cellular Biology vol 31 no 16 pp3457ndash3471 2011

[44] D V Skarra D J Arriola C A Benson and V G ThackrayldquoForkhead box O1 is a repressor of basal and GnRH-inducedFshb transcription in gonadotropesrdquo Molecular Endocrinologyvol 27 no 11 pp 1825ndash1839 2013

[45] O Mendes H-T Kim and G Stoica ldquoExpression of MMP2MMP9 and MMP3 in breast cancer brain metastasis in a ratmodelrdquo Clinical and Experimental Metastasis vol 22 no 3 pp237ndash246 2005

[46] K C Nannuru M Futakuchi M L Varney T M VincentE G Marcusson and R K Singh ldquoMatrix metalloproteinase(MMP)-13 regulates mammary tumor-induced osteolysis byactivating MMP9 and transforming growth factor-120573 signalingat the tumor-bone interfacerdquo Cancer Research vol 70 no 9 pp3494ndash3504 2010

[47] H E Barker J Chang T R Cox et al ldquoLOXL2-mediatedmatrixremodeling in metastasis and mammary gland involutionrdquoCancer Research vol 71 no 5 pp 1561ndash1572 2011

[48] N K Nickerson K S Mohammad J L Gilmore et alldquoDecreased autocrine EGFR signaling in metastatic breastcancer cells inhibits tumor growth in bone and mammary fatpadrdquo PLoS ONE vol 7 no 1 Article ID e30255 2012

[49] L Bakiri S Macho-Maschler I Custic et al ldquoFra-1AP-1 indu-ces EMT inmammary epithelial cells bymodulating Zeb12 andTGF120573 expressionrdquoCell DeathampDifferentiation vol 22 pp 336ndash350 2015


[50] A Henke O C Grace G R Ashley et al ldquoStromal expressionof decorin Semaphorin6D SPARC Sprouty1 and Tsukushi indeveloping prostate and decreased levels of decorin in prostatecancerrdquo PLoS ONE vol 7 Article ID e42516 2012

[51] J W Lee J Lee and E Y Moon ldquoHeLa human cervical cancercell migration is inhibited by treatment with dibutyryl-cAMPrdquoAnticancer Research vol 34 no 7 pp 3447ndash3455 2014

[52] S D Kim Y J Lee J S Baik et al ldquoBaicalein inhibits agonist-and tumor cell-induced platelet aggregation while suppress-ing pulmonary tumor metastasis via cAMP-mediated VASPphosphorylation along with impaired MAPKs and PI3K-Aktactivationrdquo Biochemical Pharmacology vol 92 no 2 pp 251ndash265 2014

[53] S Tao H He Q Chen and W Yue ldquoGPER mediated estradiolreduces miR-148a to promote HLA-G expression in breast can-cerrdquoBiochemical and Biophysical ResearchCommunications vol451 no 1 pp 74ndash78 2014

[54] C Strell K Lang B Niggemann K S Zaenker and F Ents-chladen ldquoSurface molecules regulating rolling and adhesionto endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interactionrdquo Cellular andMolecular Life Sciences vol 64 no 24 pp 3306ndash3316 2007

[55] I AMayer VGAbramson BD Lehmann and J A PietenpolldquoNew strategies for triple-negative breast cancermdashdecipheringthe heterogeneityrdquo Clinical Cancer Research vol 20 no 4 pp782ndash790 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


except class V contain a PSI (Plexin-semaphorin-integrin)domain immediately to the C-terminal side of the Semadomain Different classes of Semaphorins may also containadditional functional motifs

Published studies regarding functions of Semaphorins inBC have mainly focused on class 3 secreted Semaphorins andSEMA4D [14ndash17] Our recent bioinformatic analysis by usingpublic datasets reveals for the first time the potential role ofSEMA6D in regulating BC pathogenesis The extracellularregion of SEMA6D which contains the Sema and PSIdomains can be released from the cell surface to act asa secreted cytokine with unknown molecular mechanisms[26 27] Thus SEMA6D may act both locally through cell-cell contacts and more distantly through diffusion of itscleaved ectodomain The primary receptors of SEMA6D arePLXNA1 and PLXNA4 which belong to the Plexin receptorfamily The extracellular domains of Plexins interact withSemaphorins while the C-terminal tails of Plexins medi-ate intracellular signal transduction Unlike many signalingpathways (such as the TGF120573SMAD pathway) there isno ldquocanonicalrdquo intracellular transduction cascade mediatingactivities of Semaphorin-Plexin signaling Many intracellularsignaling molecules such as GTPase activating proteinsGTPGDP exchange factors and various tyrosine kinasescan be activated andor inactivated by Semaphorin-Plexinsignaling in a context-dependent manner [18ndash25]

In this study we examine the public datasets from TheCancer Genome Atlas (TCGA) National Cancer Institute(NCI) for expression of SEMA6D along with genes that inter-act with SEMA6D Other genes coregulated with SEMA6Dwere analyzed for their function pathway and potentialas copredictors for BC patientsrsquo survival We found 6-geneexpression profile that can be used as such predictorsWe alsofound that SEMA6D expression correlated with the cancerstatus of triple negative (TNBC) markers (ER PR and Her2genes) The study shows the role of SEMA6D as potentialsurvival predictor especially in TNBC patients

2 Methods

21 Datasets TheCancer GenomeAtlas (TCGA)Data Portalwas used to download breast invasive carcinoma (BRCA)samples (119899 = 1 100) The RNAseqV2 level 3 data whichincludes fragments per kilobase of exon per million frag-ments mapped- (FPKM-) normalized gene level data wereused before statistics In addition idf and sdrf files were alsodownloaded for sample mapping and annotation Clinicaloutcomes data were downloaded for correlation and survivalanalysis

22 Gene Expression Data Analysis and Annotation Gene-level normalized expression data were used in PartekGenomic Suite (PGS St Louis MO) for additional normal-ization statistics and annotation The analysis of variance(ANOVA) methods were used for group comparisons Falsediscovery rate (FDR) correction (Benjamini-Hochbergmeth-ods) was applied for multiple hypothesis testing purposeOther statistical tools such as SAS (Cary NC) and IngenuityPathway Analysis (IPA Redwood City CA) were used for

pathway analysis and building gene-gene interaction net-work Heatmap was generated by using hierarchical cluster-ing methods after z-normalization

23 Survival Analysis A total of 140 patients with clinicaloutcomes data available (survival status months of sur-vival demographics and ER PR and HER2 status etc)were included in the analysis Among significant genesafter SEMA6D-high versus SEMA6D-low expression com-parisons we selected top 20 genes with the highest or lowestexpression levels to correlate with clinical outcomes Loga-rithm 2 based transformation of each gene was performedprior to any analysis The correlation among these 20 geneswas evaluated using Pearson correlation coefficient and sum-mary statistics were presented including mean with standarddeviation median and range Associations between level ofgenes and overall survival (OS) were assessed with Kaplan-Meier (K-M) curves and log-rank tests Each gene wasdichotomized as above or belowmedian level of expression inthe survival analysis Significant association was determinedat 5 type I error level Multiple comparisons were notexplicitly controlled for due to the small sample size andexploratory nature of the analysis

3 Results and Discussion

Semaphorins including members in subclass 3 andSEMA4D have emerged as critical signaling moleculesin regulating BC pathology [14ndash17] The potential roles ofother members of the Semaphorin family in BC have notbeen well addressed in the literature Our ongoing studiessuggest that SEMA6D plays a critical role in mediatingBone Morphogenetic Protein (BMP) signaling to regulateepithelial-mesenchymal-transition (EMT) by endocardialcells in developing hearts (Kai Jiao et alrsquos manuscript inpreparation) EMT is an essential step for initiatingmetastasisin BC and other cancersWe thus decided to apply a bioinfor-matic approach to examine the potential role of SEMA6Din BC tumorigenesis and progression using publically avail-able datasets After testing a few smaller datasets form GeneOntology Omnibus (GEO) we chose to use a breast invasivecarcinoma (BRCA) dataset from TCGA as it represents amajor public data source with clinical outcomes informationsuch as overall survival after diagnosis We divided all sam-ples (119899 = 1 100) into three roughly equal sized groups basedon SEMA6D expression (high medium and low) The com-parison of SEMA6D-high versus SEMA6D-low expressiongroup will reveal genes that are coregulated with SEMA6D

Gene Expression Profile by Principle Components Analysis(PCA) and Hierarchical Clustering To examine overall geneexpression profile and sample similarities we perform thePCA analysis of all samples As showed in Figure 1 thePCA showed a clear separation among SEMA6D-high (H)SEMA6D-medium (M) and SEMA6D-low (L) groups Thisindicates different gene expression profiles among the threegroups

Based on the genes that are differentially expressed inSEMA6D-high versus SEMA6D-low expression groups we


PC

2 5

28

PC 1 795

PC 3 509

PCA mapping (183)

HLM

PC CC 3 509PCC
















Mitosis 1854 890119864 minus 09




















500000minus500

L

L

L

L

LLL

L

LL

L

L

L

L

L

L

L

L

L

L

L

H

H

H

H

M

M

MM

M

M

M

MMM

M

M

M

M

M

M

M



58 255 2357






Plasma membrane

Cytoplasm

Nucleus

PLXNA1

PLXNA4

5428

16518

AKT2

AKT1

PI3K (complex)

AKT3

FOSL1

SRF FOS

FOXO1

2711

4908

Sos

ABL1

SEMA6D Enah









Foldchange

Fold(description)


H downversus L





















ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79


SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55


VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30



KRT19 minus026 277119864 minus 18


COL3A1 017 102119864 minus 08



VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209






CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects


70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian








08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PC

2 5

28

PC 1 795

PC 3 509

PCA mapping (183)

HLM

PC CC 3 509PCC
















Mitosis 1854 890119864 minus 09




















500000minus500

L

L

L

L

LLL

L

LL

L

L

L

L

L

L

L

L

L

L

L

H

H

H

H

M

M

MM

M

M

M

MMM

M

M

M

M

M

M

M



58 255 2357






Plasma membrane

Cytoplasm

Nucleus

PLXNA1

PLXNA4

5428

16518

AKT2

AKT1

PI3K (complex)

AKT3

FOSL1

SRF FOS

FOXO1

2711

4908

Sos

ABL1

SEMA6D Enah









Foldchange

Fold(description)


H downversus L





















ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79


SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55


VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30



KRT19 minus026 277119864 minus 18


COL3A1 017 102119864 minus 08



VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209






CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects


70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian








08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















500000minus500

L

L

L

L

LLL

L

LL

L

L

L

L

L

L

L

L

L

L

L

H

H

H

H

M

M

MM

M

M

M

MMM

M

M

M

M

M

M

M



58 255 2357






Plasma membrane

Cytoplasm

Nucleus

PLXNA1

PLXNA4

5428

16518

AKT2

AKT1

PI3K (complex)

AKT3

FOSL1

SRF FOS

FOXO1

2711

4908

Sos

ABL1

SEMA6D Enah









Foldchange

Fold(description)


H downversus L





















ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79


SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55


VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30



KRT19 minus026 277119864 minus 18


COL3A1 017 102119864 minus 08



VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209






CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects


70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian








08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Plasma membrane

Cytoplasm

Nucleus

PLXNA1

PLXNA4

5428

16518

AKT2

AKT1

PI3K (complex)

AKT3

FOSL1

SRF FOS

FOXO1

2711

4908

Sos

ABL1

SEMA6D Enah









Foldchange

Fold(description)


H downversus L





















ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79


SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55


VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30



KRT19 minus026 277119864 minus 18


COL3A1 017 102119864 minus 08



VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209






CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects


70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian








08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Foldchange

Fold(description)


H downversus L





















ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79


SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55


VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30



KRT19 minus026 277119864 minus 18


COL3A1 017 102119864 minus 08



VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209






CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects


70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian








08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















ZEB1 055 138119864 minus 87

TCF4 053 532119864 minus 82

AHNAK 052 708119864 minus 79


SNAI2 047 266119864 minus 62

ZEB2 046 153119864 minus 59

MITF 045 523119864 minus 55


VEGFC 038 927119864 minus 39

MMP2 033 547119864 minus 30



KRT19 minus026 277119864 minus 18


COL3A1 017 102119864 minus 08



VEGFB 011 0000235

KRT7 minus009 0002199

BMP7 007 0015785

DSC2 minus004 0188209






CLEC9A 127 212 193 236 minus187 573 00308

COL4A6 139 565 267 579 018 1077 00564C10orf107 134 250 203 267 minus202 694 00019

DONSON 140 799 091 787 636 1046 00397

CHAC1 140 496 156 483 119 886 00003

TUBA1C 140 1161 099 1174 939 1451 00162



08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi

lity

Log-rank p = 00156

MEDlog2SEMA6D

gtMedian

leMediangtMedian

Numberof subjects


70

70

70

70

0

0

29 (22-43)53 (43-60)

OS_Yr

leMedian








08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















08

06

04

02

0000 25 50 75 100 125

Surv

ival

pro

babi


OS_Yr




(95 CI)

0

0

0

0

32 (23-50)

51 (38-60)

24 (07-43)

81 (46-95)

58

64

12

6


4 Conclusions


Abbreviations






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article sema6d expression and patient survival in...

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