gain-of-function of mutant p53: mutant p53 enhances cancer progression by inhibiting klf17...

Original Article

Gain-of-function of mutant p53 mutant p53 enhances cancerprogression by inhibiting KLF17 expression in invasive breastcarcinoma cellsAmjad Ali a Abdus Saboor Shah a Ayaz Ahmad bca Institute of Biomedical Sciences School of Life Sciences East China Normal University 500 Dongchuan Road Shanghai 200241 Chinab Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing 100101 Chinac Department of Biotechnology Abdul Wali Khan University Mardan 23200 Pakistan

A R T I C L E I N F O

Article historyReceived 23 January 2014Received in revised form 4 April 2014Accepted 30 July 2014

KeywordsMetastasisKLF17Mutant p53Breast cancerChemotherapyInvasion

A B S T R A C T

Kruppel-like-factor 17 (KLF17) is a negative regulator of metastasis and epithelial-mesenchymal-transition (EMT) However its expression is downregulated in metastatic breast cancer that contains p53mutations Here we show that mutant-p53 plays a key role to suppress KLF17 and thereby enhancescancer progression which defines novel gain-of-function (GOF) of mutant-p53 Mutant-p53 interacts withKLF17 and antagonizes KLF17 mediated EMT genes transcription Depletion of KLF17 promotes cell vi-ability decreases apoptosis and induces drug resistance in metastatic breast cancer cells KLF17 suppressescell migration and invasion by decreasing CD44 PAI-1 and Cyclin-D1 expressions Taken together ourresults show that KLF17 is important for the suppression of metastasis and could be a potential thera-peutic target during chemotherapy

copy 2014 Elsevier Ireland Ltd All rights reserved

Introduction

Tumor suppressor TP53 is the most frequently mutated gene inhuman cancer including breast cancer [12] Many of these muta-tions give rise to mutant p53 proteins that are highly expressed incancer cells and gain new functions that contribute to tumor pro-gression [34] Most missense mutations in the TP53 gene occursin the DNA binding domain of p53 [5] To date mutant p53 pro-teins have been considered in two different groups (1) DNA contactmutants affecting amino acids that contact DNA without alteringthe conformation of p53 protein such as R273H R248W and R282Wand (2) conformational mutants disrupting the three-dimensionalstructure of p53 protein such as R157H R249S and R280K [67] Ac-cumulating data show that mice expressing mutant p53 display moreaggressive tumor profile and metastatic potential than p53 wild-type or p53 null mice [38] Xenograft mouse models and in vitrostudy have provided strong evidence that mutant p53 proteins driveenhanced invasion and motility [910] Mutant p53 exerts its gain-of-function through different mechanisms such as (1) mutant

p53 interacts with and inactivates the proteins involved in tumorinhibition for instance p73 and p63 [610] (2) Mutant p53 can bindto DNA without genotoxic stress and drive the expression of itsown set of target genes such as GRO-1 MYC CXCL1 ID2 and MAP2K3[611] (3) Mutant p53 is involved in the formation of transcrip-tionally competent complexes via proteinndashprotein interactionthrough which the expression of its target genes is regulatedsuch as NF-Y SREBP and VDR [612ndash14] In addition mutant p53 hasbeen demonstrated to transactivate the genes that can promote pro-liferation and migration of cancer cells inhibit apoptosis andenhance drug resistance [1516] Despite correlation of the expres-sion of p53 mutants and poor prognosis of human breast cancerpatients the role of mutant p53 proteins in driving breast cancermetastasis and epithelial-mesenchymal-transition remains unclear[1617]

Kruppel-like-factor 17 (KLF17) is a tumor suppressor transcrip-tion factor which inhibits metastasis and EMT [18ndash20] KLF17 bindsto its target gene promoters via CACCC boxes and regulates theirexpression [18ndash20] KLF17 mainly exerts its tumor suppressor func-tion by acting on the promoters of EMT-related genes such as id1E-cadherin ZO-1 vimentin and fibronectin [18ndash20] Recent studieshave shown that KLF17 expression is negatively regulated by someoncogenes such as DJ-1 and microRNA-9 to enhance cancer cell in-vasion and migration [1920] Studies have shown that invasive breastcancer cells and breast cancer tissues with lymph node metastasis

Corresponding author Tel +86-15921906215 fax + 86-21-64154900E-mail address amjad_486yahoocom (A Ali)

Corresponding author Tel +92-3009598201E-mail address ahdayazb5awkumedupk (A Ahmad)

httpdxdoiorg101016jcanlet2014070450304-3835copy 2014 Elsevier Ireland Ltd All rights reserved

Cancer Letters 354 (2014) 87ndash96

Contents lists available at ScienceDirect

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express low level of KLF17 [182122] Moreover knockdown of KLF17enhances metastasis and EMT in vivo [1819] In contrast forcedexpression of KLF17 inhibits proliferation and growth of cancer cells[2324] Decrease expression of KLF17 is an independent indicatorof poor prognosis for patients with lung adenocarcinomaheptocellular and breast carcinoma [181924] Patients with lowerexpression of KLF17 had greater tumor size later pathological stageand poor prognosis [2324] Although lines of evidence suggest thatKLF17 inhibits cancer progression the signaling pathways control-ling KLF17 function largely remains unknown

Here we show that the expression of metastatic suppressor KLF17is controlled by mutant p53 proteins to potentiate cancer progres-sion in metastatic breast carcinoma Mutant p53 exerts its novel gain-of-function (GOF) ability to inhibit KLF17 expression KLF17 lowexpression in metastatic breast cancer cells can be specifically drivenby mutant p53 that is recruited to upstream of KLF17 promoter Fur-thermore endogenous mutant p53 proteins such as p53-R280K andp53-R282W have the ability to form a complex with KLF17 and an-tagonize KLF17 mediated EMT genes transcription Forced expressionof KLF17 decreases drug resistance in response to chemotherapyIntroduction of KLF17 to mutant p53 containing breast cancer cellssignificantly inhibited migration and invasion by repressing anetwork of oncogenes including PAI-1 CD44 and Cyclin-D1 Our

finding demonstrates that repression of KLF17 by mutant p53 hasa key role in enhancing cancer progression

Materials and methods

Plasmids and transfection

pcDNA31-R175H pcDNA31-R273H pcDNA31-p53 plasmids were described inour previous study [25] H1299 MDA-MB-1386 and MDA-MB-231 cancer cell lineswere transfected with Lipofectamine 2000 following manufacturerrsquos protocol(Invitrogen)

Antibodies

The following antibodies were used in Western Blot and ChIP experiments anti-p53 (DO-1 Santa Cruz) anti-KLF17 (Abcam) and anti-GAPDH (Santa Cruz)

Cell culture and treatments

H1299 MDA-MB-1386 MDA-MB231 and MCF-7 cells were described previouslyin our study [25] For cell treatments we used (05 μM) Adriamycin (Sigma-Aldrich)

Electrophoretic mobility shift assay (EMSA)

EMSA were performed as described previously [25] Briefly 2 μg of nuclearextract or different concentration of purified proteins was incubated with

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Fig 1 Mutant p53 negatively regulates KLF17 in metastatic breast cancer cells (A and B) MDA-MB-231 and MDA-MB-1386 cells were transfected with control siRNA orsiRNA targeting against p53 (20 nM) for 48 hr Total RNA was extracted and subjected to quantitative RT-PCR (qRT-PCR) analysis The average was calculated based on threeindependent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005 P lt 0005) (C and D) MDA-MB-231 and MDA-MB-1386 cells were transfected with controlsiRNA or siRNA targeting against p53 (20 nM) for 72 hr and subjected to immunoblot analyses with indicated antibodies (E) MCF-7 cells were transfected with control siRNAor siRNA targeting against p53 (20 nM) for 72 hr and subjected to immunoblot analyses with indicated antibodies

88 A Ali et alCancer Letters 354 (2014) 87ndash96

32P-radiolabeled-probes in 20 μl of EMSA reaction buffer (2 μg of poly (dI-dC) 20 mMHEPES (pH 79) 1 mM MgCl2 40 mM KCl 01 mM EDTA 1 mM DTT and 12 glyc-erol) To perform the competition assay an excess of unlabeled competitorrsquos oligowas added to the EMSA reaction mixture Protein-DNA complexes were resolved in5 polyacrylamide gels containing 05x TBE and exposed to phosphoimager (Bio-Rad) The following probe sequences were used for EMSA

ZO-1 probe forward 5-TGGACTGCACGTGTTTACACCCTTTTCTGCCCAACTTTGA-3ZO-1 probe reverse 5-TCAAAGTTGGGCAGAAAAGGGTGTAAACACGTGCAGTCCA-3ZO-1mutant probe forward 5-TGGACTGCACGTGTTTAGATACTTTTCTGCCCAACTTTGA-3ZO-1mutant probe revers5-TCAAAGTTGGGCAGAAAAGTATCTAAACACGTGCAGTCCA-3

Chromatin immunoprecipitation assay (ChIP)

ChIP was performed as described previously [25] Briefly after being trans-fected with flag-KLF17 nuclear proteins were cross linked to genomic DNA by addingformaldehyde for 10 min directly to the medium to a final concentration of 1 Cross-linking was stopped by adding glycine to a final concentration of 0125 M andincubating for 5 min at room temperature on a rocking platform The medium wasremoved and the cells were washed twice with ice cold phosphate buffered saline(PBS) After centrifugation the cell pellets were resuspended in lysis buffer Cellu-lar debris was removed by centrifugation and the lysates were diluted 110 in ChIPdilution buffer The samples were centrifuged and the recovered chromatin solu-tions were incubated with 3ndash5 μg of indicated antibodies overnight at 4 degC withrotation The immuno-complexes were collected with 60 μl of protein A agarose slurryfor 2 h at 4 degC with rotation PCR-amplification of the genomic-fragments was per-formed with specific primers flanking putative binding-sites on the KLF17 promoterand on EMT target gene promoters

KLF17 luciferase reporter constructs

DNA-fragments containing KLF17genomic sequences were amplified from 293Tcell genomic DNA using the polymerase chain reaction and primers derived fromhuman genomic KLF17 and ligated into kpn1xhol sites of the promoterless pGL3-

Basic (Promega) vector and was named as pGL3-KLF17-luc Different deletionconstructs of KLF17 promoter were generated from 2 kb KLF17 promoter and ligatedinto kpn1xhol sites of pGL3-Basic vector

Luciferase assay

Luciferase assay was performed as described previously [25]

RNA interference

Cells were cultured to 30 confluence For each well in a 6-well culture dish20 nM of KLF17p53 siRNAs or appropriate negative controls siRNAs were trans-fected into cells using Lipofectamine 2000 (Invitrogen) following manufacturerrsquosprotocol Cells were incubated at 37 degC in a CO2 incubator and 6ndash8 hr later 10 serumgrowth medium was added to the transfection mixture Cell-extracts were assayedby Western blot for KLF17p53 protein expression at 72 hr post transfection whilefor mRNA expression was at 48 hr after transfection

RT-PCR

RT-PCR was performed as described previously [25] Total RNA from cells wasisolated using TRIZOL (Invitrogen) following manufacturerrsquos protocol Each exper-iment was performed in duplicates and repeated thrice

MTT assay

MTT assay was performed as described previously [25] Briefly cell viability wasassessed with a 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium-bromide (MTT)assay in replicates Cells were seeded in 96-well plate at 25 times 103 cellswell and in-cubated in 10 FBS supplemented with DMEM for 24 h After that cells were treatedwith Adriamycin for indicated time points Controls received DMSO vehicle at a con-centration equal to that in drug-treated cells After that drug-containing medium

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Fig 2 Mutant p53 represses KLF17 promoter in cancer cells (A) p53 null H1299 lung cancer cells were co-transfected with KLF17 reporter construct (2 μg) in combinationwith different concentrations of p53-R175H for 24 hr prior to lysis and analyzed for luciferase-activity Data are representative of three independent experiments (mean plusmn sd)(Two-tailed Studentrsquos t-test P lt 005 P lt 0005) (B) p53 null H1299 lung cancer cells were co-transfected with KLF17 reporter construct (2 μg) in combination with p53-R175H (50 ng) p53-R273H (50 ng) p53-R280K (50 ng) and p53-R282W (50 ng) for 24 hr prior to lysis and analyzed for luciferase-activity Data are representative of threeindependent experiments (mean plusmn sd) (Two-tailed Studentrsquos t-test P lt 005) (C) Schematic map of different deletion constructs of KLF17 (D) Different luciferase con-structs of KLF17 (each 2 μg) were co-transfected with p53-R175H (50 ng) for 24 hr and analyzed by luciferase assay Data are representative of three independent experimentswith mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005)

89A Ali et alCancer Letters 354 (2014) 87ndash96

was replaced with 200 μL of 10 FBS supplemented with DMEM containing 05 mgmL MTT and cells were incubated in the CO2 incubator at 37 degC for 2 h and absorbance(490 nm) was measured and analyzed

Cell cycle analysis

Cell-cycle analysis was carried out estimating DNA-contents with flow cytometryas described previously [25] Cells were fixed in ice cold 70 ethanol incubated over-night at ndash20 degC and stained with propidium-iodide (PI)Triton X-100 containingRNaseA-solution for 15 minutes at 37 degC Cell-cycle analysis was performed usingBD CantoII cell-analyzer

Cell migration

Cell culture inserts with a pore size of 8 μm were pre-coated by adding 10 μgmL collagen I in PBS to the upper chamber at room temperature for 2 hr At the onsetof each experiment the cells were detached with Versene and resuspended as singlecells in serum-free DMEM For the migration experiments we took 25000 cells in05 mL and diluted them in serum-low DMEM (supplemented with 2 FBS) Cellswere added to the upper chamber and the lower chamber was filled with 070 mLDMEM supplemented with 10 FBS The cells were allowed to migrate for 24 h at37 degC in a humidified atmosphere of 5 CO2 The experiment was terminated by dis-carding the medium and fixing the cells in the filter with 4 paraformaldehyde for10 min Non-invading cells on the upper side of the insert were removed by a cotton-tipped applicator Staining of the cells on the bottom of the membrane was performedwith DAPI (300 nM in PBS) for 5 min at room temperature and washed with PBSMembranes were excised from the inserts and mounted on slides using Dako Flu-orescent Mounting Medium Cells were either counted manually or with the helpof the Image J software

Cell invasion

Cell invasion experiments were performed in BD Matrigeltrade invasion cham-bers (BD Biosciences) with a membrane pore diameter of 8 μm For the invasion

experiments we used 50000 MDA-MB-1386 cells per well suspended in 05 mLserum-low DMEM (supplemented with 2 FBS) The rest of the protocol was iden-tical to that described in the migration assay

Results

Mutant p53 suppresses KLF17 in metastatic breast cancer cells

Inactivation or mutations of p53 signaling contributes to cancerdevelopment and progression which is called gain-of-function(GOF) of mutant p53 [157] Low expression of KLF17 has been de-tected in metastatic breast cancer cells but its molecular mecha-nism remains elusive These hints prompted us to examine theregulation of KLF17 in metastasis breast cancer cells To thisend we used advance metastatic breast cancer cells such asMDA-MB-231 and MDA-MB-1386 that contain naturallyharboring mutant p53 (p53-R280K) and (p53-R282W) proteinsrespectively

Depletion of mutant p53 in these two cell lines increased themRNA and protein levels of KLF17 (Fig 1AndashD) In contrast silenc-ing of p53 in non-metastatic MCF-7 breast cancer cells which expresswild-type p53 decreased the mRNA and protein levels of KLF17(Fig 1E and supplementary Fig S1A) Next we compared the KLF17expression in non-metastatic and metastatic breast cancer cellswhich express wild-type p53 and mutant p53 respectively We ob-served higher level of KLF17 in non-metastatic breast cancer cellscompared with metastatic breast cancer cells (SupplementaryFig S1B and C) In addition reporter assay showed that mutantp53 inhibits KLF17 transcription in comparison with WT-p53(Supplementary Fig S1D) These results suggest that mutant p53

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Fig 3 Mutant p53 binds toKLF17 promoter in breast cancer cells (A) Schematic representation of ChIP primers from KLF17 promoter (BndashE) MDA-MB-1386 and MDA-MB-231 cells were transfected with control siRNA or siRNA targeting against p53 (20 nM) for 72 hr and subjected to ChIP analysis with p53 antibody (C and E) The average wascalculated based on three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005)


proteins play a key role to repress KLF17 expression in metastaticbreast carcinoma

KLF17 is a direct target of mutant p53 in cancer cells

Next we aimed to examine the transcriptional regulation ofKLF17 by mutant p53 proteins We co-transfected KLF17-Lucconstruct with different combinations of mutant p53-R175Hinto H1299 (p53 null) cells Mutant p53-R175H was able to repressKLF17 transcription in H1299 cells (Fig 2A) Next we observed re-pression of KLF17 reporter by both DNA conformation (p53-R175H and p53-R280K) and DNA contact (p53-R273H and p53-R282W) mutants suggesting that KLF17 is a target of differentmutant p53 (Fig 2B) Deletion analysis of KLF17-Luc constructs re-vealed that the region between (ndash2000 to ndash1566) is responsive tomutant p53 but not the region beyond (ndash1566+1) (Fig 2C andD) These data suggest that mutant p53 responsive region liesbetween (ndash2000 to ndash1566)

Mutant p53 recruits to KLF17 promoter in metastaticbreast carcinoma

Mutant p53 binds with its target gene promoter without any ex-ternal stimuli such as GRO-1 ID2 ID4 CD95 EGR1 and MSP [2627]Therefore we examined the physical association of mutant p53 pro-teins with KLF17 promoter in metastatic breast cancer cells ChIPanalyses disclose the recruitment of mutant p53 proteins to KLF17

promoter in metastasis breast cancer cells (Fig 3AndashE) In contrastno binding of mutant p53 was detected to the further upstreamregion of KLF17 promoter (Fig 3B and D lower panels) These datasuggest that endogenous mutant p53 proteins have the ability torecruit to KLF17 promoter

Mutant p53 antagonizes KLF17 mediated EMT genes expression inmetastatic breast carcinoma

Our results indicate that KLF17 is a target of mutant p53 pro-teins in advance breast cancer cells KLF17 is a transcription factorthat binds to the promoters of EMT target genes to inhibit metas-tasis [182829] Therefore we investigated the effect of mutant p53proteins on KLF17 mediated EMT genes transcription KLF17 inhib-its metastasis and tumor growth by negatively regulating id1vimentin and fibronectin expression while inducing the ZO-1 ande-cadherin transcript [181923] To this end mutant p53 depletedand control metastatic breast cancer cell lines were left untransfectedor transfected with KLF17 expression vector and qRT-PCR was per-formed to check the mRNA level of the KLF17 target genes Transcriptanalysis of these target genes revealed that the tumor suppressiveability of KLF17 was lower in mutant p53 containing breast cancercells (Fig 4AndashH and Supplementary Fig S1A and B) In contrast de-pletion of mutant p53 enhanced the metastasis suppressor abilityof KLF17 to EMT target genes in breast cancer cells (Fig 4AndashH andSupplementary Fig S2A and B) Taken together mutant p53 revertsthe expression of KLF17 target genes in metastatic breast cancer cells

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Fig 4 Knockdown of mutant p53 enhances KLF17 mediated target genes expressions (AndashH) MDA-MB-231 and MDA-MB-1386 breast cancer cells were transfected withcontrol siRNA or siRNA targeting against mutant p53 (20 nM) for 48 hr and then transfected with control vector or expression plasmid encoding KLF17 for 24 hr Total RNAwas extracted and qRT-PCR was performed The average was calculated based on three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005)


Mutant p53 prevents recruitment of KLF17 to EMTtarget gene promoters

To further gain insight on how mutant p53 decreases the tumorsuppressor ability of KLF17 to enhance metastasis we tested the in-teraction between mutant p53 proteins and KLF17 byimmunoprecipitation (IP) in MDA-MB-231 and MDA-MB-1386 cellsWe observed that mutant p53 co-immunoprecipitated with KLF17in both these cell lines (Fig 5A) These data suggest that mutantp53 forms a complex with KLF17 and decreases the metastatic sup-pressor function of KLF17 in these cell lines Moreover we performedChIP assay on KLF17 mediated EMT-related genes in the presenceor absence of mutant p53 We selected Vimentin and ZO-1 promot-ers which are known targets of KLF17 [19] ChIP analysis showedreduced recruitment of KLF17 to Vimentin and ZO-1 promoters inmutant p53 containing breast cancer cells (Fig 5BndashE) In contrast si-lencing of mutant p53 enhanced KLF17 physical association with EMTtarget gene promoters (Fig 5BndashE) Next we performed EMSA to observethe formation of KLF17-DNA complex EMSA analysis revealedthe formation of stronger KLF17-DNA complex in mutant p53 de-pleted breast cancer cell transfected with KLF17 (Fig 5F lane 3)Conversely the formation of KLF17-DNA complex is reduced in mutant

p53 containing cell lines transfected with KLF17 (Fig 5F comparelane 6 and 7 with lane 3) Taken together these results indicate thatmutant p53 decrease the recruitment of KLF17 to EMT target genepromoters

KLF17 suppresses cancer progression in advance metastatic breastcancer cells

To address the biological impact of KLF17 on metastatic breastcancer cells we depleted the expression of KLF17 in MDA-MB-231and MDA-MB-1386 cell lines MTT assay showed that silencing of KLF17increased cell viability in these metastatic breast carcinoma whichwas comparable with control cells (Fig 6A and B) In contrast forcedexpression of KLF17 in mutant p53 containing breast cancer cellscaused inhibition of cell growth (Fig 6C) Moreover knockdown ofKLF17 decreased drug sensitivity of MDA-MB-1386 breast cancer cellsin response to Adriamycin as compared with control cells (Fig 6D andSupplementary Fig S3A) Importantly FACS analyses showed that de-pletion of KLF17 decreased apoptotic level of MDA-MB-1386 breastcancer cells treated with Adriamycin (Fig 6E) Taken together knock-down of KLF17 is associated with cancer progression in metastaticbreast cancer cells

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Fig 5 Mutant p53 reduces recruitment of KLF17 to EMT target gene promoters (A) Mutant p53 co-immunoprecipitates with KLF17 in whole-cell lysates prepared fromMDA-MB-231 and MDA-MB-1386 cells (BndashE) MDA-MB-231 and MDA-MB-1386 breast cancer cells were transfected with control siRNA or siRNA targeting against mutantp53 (20 nM) for 48 hr and then transfected with expression plasmid encoding KLF17 for 24 hr ChIP was performed with antibody specific for KLF17 The average was cal-culated based on three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005) (F) MDA-MB-231 breast cancer cells were transfected with controlsiRNA or siRNA targeting against mutant p53 (20 nM) for 48 hr and then transfected with expression plasmid encoding KLF17 for 24 hr Radioactive EMSA was performedon the oligo-probe derived from ZO-1 promoter (compare lane 3 with lanes 6 and 7)


KLF17 inhibits metastatic breast cancer cell migration and invasion

It is well established that mutant p53 positively regulate cancercell migration [1ndash6] As our results indicate that mutant p53 in-hibits KLF17 in breast cancer cells therefore we next performedTrans-well migration and invasion assays with metastatic breastcancer cells in the absence or presence of KLF17 MDA-MB-1386 andMDA-MB-231 breast cancer cells depleted with KLF17 migrated moreas compared with control cells (Fig 7A and D) Moreover forced ex-pression of KLF17 in MDA-MB-1386 and MDA-MB-231 breast cancercells inhibited migration in contrast with control cells transfectedwith empty vector (Fig 7B and E) We obtained similar results ofKLF17 on metastatic breast cancer cell invasion as the penetra-tion of the cells through the matrigel was significantly increasedupon depletion of KLF17 when compared to control cells (Fig 7Cand F) These results indicate that KLF17 restricts migration and in-vasion of advance metastatic breast cancer cells

KLF17 represses CD44 Cyclin-D1 and PAI-1 to inhibit metastasis inbreast cancer cells

Our next goal was to identify KLF17 novel target genes that couldbe linked to breast cancer cell migration and invasion Intrigu-ingly we found that CD44 Cyclin-D1 and PAI-1 were negativelyregulated by KLF17 in breast cancer cells CD44 Cyclin-D1 and PAI-1are involved in breast cancer progression [30ndash32] Silencing of KLF17enhanced the transcript of these target genes (Fig 8AndashD) whileoverexpression of KLF17 inhibited the mRNA level of these genes

in MDA-MB-231 breast cancer cells (Supplementary Fig S4AndashC)KLF17 is a tumor suppressor transcription factor and it binds to itstarget gene promoters through CACCC boxes [181929] We foundputative KLF17 binding sequence in these target gene promoters(Fig 8E) ChIP analyses indicated that KLF17 recruited to CD44Cyclin-D1 and PAI-1 promoters which suggests that these targetgenes are directly regulated by KLF17 in breast cancer cells(Fig 8FndashH) Taken together our data show that KLF17 targets somekey genes in advance metastatic breast cancer cells to restrict me-tastasis and EMT

Discussion

Recent studies have shown that KLF17 inhibits metastasis andEMT in cancer cells [18ndash21] However the molecular mechanismthrough which KLF17 tumor suppressive ability is lost largely remainsunknown We found decreased KLF17 expression in highly inva-sive breast cancer cells as compared with non-metastatic cells Insearch for the mechanism we identified that KLF17 is a novel targetgene of mutant p53 Mutant p53 is an oncogenic transcription factorwhich promotes tumor growth and metastasis aggressively by de-creasing expression of tumor suppressor genes or inactivates themvia multiple mechanisms [625] Our results indicate that mutantp53 represses KLF17 expression in invasive breast cancer cells whichdefines a novel gain-of-function of mutant p53 In addition en-dogenous mutant p53 from invasive breast carcinoma binds to aspecific region of KLF17 promoter Moreover transcriptional reg-ulation of KLF17 by DNA contacts and conformational p53 mutants

CTL-siRNA

KLF17-siRNACTL-siRNA+Adriamycin

KLF17-siRNA+Adriamycin

A B C

D

CTL-siRNAKLF17-siRNA

0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72

MDA-MB-1386 breast cancer cells

0

01

02

03

04

05

06

07CTL-siRNAKLF17-siRNA

hr 0 24 48 72

OD

valu

e

Adriamycin


0

10

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30

40

50

60

a

popt

osis


0

01

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06

hr 0 24 48Flag-KLF17

OD

valu

e

E


hr 0 24 48 72

OD

valu

e

0

01

02

03

04

05


Fig 6 Knockdown of KLF17 induces cancer progression in invasive breast cancer cells (A and B) MDA-MB-1386 and MDA-MB-231 breast cancer cells were transfectedwith control siRNA or siRNA targeting against KLF17 (20 nM) for indicated time points and MTT assay was performed Data are representative of three independent experi-ments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005 P lt 0005) (Two way ANOVA P lt 0005) (C) MDA-MB-1386 cells were transfected with control vector orexpression plasmid encoding KLF17 for indicated time periods and MTT assay was performed Data are representative of three independent experiments with mean plusmn sd(Two-tailed Studentrsquos t-test P lt 005 P lt 0005) (D) MDA-MB-1386 cells was transfected with control siRNA or siRNA targeting against KLF17 (20 nM) and then left un-treated or treated with Adriamycin (1 μM) for indicated time points and MTT assay was performed Data are representative of three independent experiments with mean plusmn sd(Two-tailed Studentrsquos t-test P lt 005 P lt 0005) (Two way ANOVA P lt 0005) (E) MDA-MB-1386 cells were transfected with control siRNA or siRNA targeting againstKLF17 (20 nM) for 48 hr and then left untreated or treated with Adriamycin (1 μM) for 24 hr and FACS analysis was performed to detect the apoptotic level Data are rep-resentative of three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005)


indicate that KLF17 is an important target of mutant p53 duringcancer progression

Our results indicate that mutant p53 antagonizes KLF17 medi-ated EMT target genes transcription in advance metastatic breastcancer cells This ability of mutant p53 defines its novel gain-of-function Accumulating data have shown that mutant p53 inhibitsthe tumor suppressive function of target genes via proteinndashprotein interaction such as p63 and p73 [6710] Our data indicatethat mutant p53 physically interacts with KLF17 and results in theformation of a complex in advance breast cancer cells The netoutcome of this interaction is to inactivate the KLF17 and ultimate-ly lead to decreased recruitment of KLF17 to EMT target genepromoters (Fig 9A) Moreover mutant p53 also affects the DNAbinding ability of KLF17 and nearly abolishes the formation of KLF17-DNA complex

We have shown that inhibition of KLF17 by mutant p53 has func-tional outcomes Silencing of KLF17 in MDA-MB-231 and MDA-MB-1386 cells enhanced the proliferation ability of these cell linesIn addition introduction of KLF17 to mutant p53 containing cellsenhanced apoptosis in advance breast carcinoma Moreover KLF17enhances drug sensitivity in response to chemotherapy in inva-sive breast carcinoma

Our data also indicate the important role of KLF17 in meta-static breast cancer cell migration and invasion Silencing of KLF17is associated with more cell migration and invasion of MDA-MB-

231 and MDA-MB-1386 cells In contrast forced expression of KLF17decreases the migration and invasion ability of breast cancer cellsIn search for the mechanism on how KLF17 inhibits breast cancercell invasion and migration we identified new target genes of KLF17in breast cancer cells We show that KLF17 negatively regulates theexpression of CD44 Cyclin-D1 and PAI-1 and directly binds to thepromoters of these target genes in breast cancer cells to restrictmetastasis

The decreased expression of WNT signaling target genes suchas Cyclin-D1 and CD44 by KLF17 in advance breast cancer cells sug-gests that KLF17 may be an important negative regulator of the WNTsignaling pathway that is involved in many biological processes in-cluding cancer progression [33] However further definition andinvestigation of KLF17 regulated genes are important to under-stand the signaling pathways through which KLF17 exerts its tumorsuppressive activity in cancer cells

In conclusion our study indicates a novel gain-of-function ofmutant p53 in invasive breast cancer cells Our results indicate howmutant p53 enhances cancer progression by suppressing KLF17 ex-pression in invasive breast carcinoma In addition forced expressionof KLF17 inhibits cancer progression in advance breast cancer cellssuch as MDA-MB-231 and MDA-MB-1386 that contain mutant p53proteins Our data indicate that KLF17 plays a key role in the re-striction of cancer growth in metastatic breast cancer cells and couldbe an important chemotherapeutic target

A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386

siRNA-CTL siRNA-KLF17

0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386

Flag-KLF17Flag-Vector

0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

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12

MDA-MB-231


Mig

ratio

n in

dex

0

01

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05

06

07

08

09

1

0

02

04

06

08

1

12

14

Fig 7 Silencing of KLF17 enhances breast cancer cell migration and invasion (A) MDA-MB-1386 cells were transfected with control siRNA or siRNA targeting against KLF17(20 nM) and cell migration assay was performed Data are representative of three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005) (B) MDA-MB-1386 cells were transfected with flag-vector or vector encoding KLF17 plasmid for 24 hr and cell migration assay was performed Data are representative of three independentexperiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 0005) (C) MDA-MB-1386 cells were transfected with control siRNA or siRNA targeting against KLF17 (20 nM)and cell invasion assay was performed Data are representative of three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005) (D) MDA-MB-231 cells were transfected with control siRNA or siRNA targeting against KLF17 (20 nM) and cell migration assay was performed Data are representative of three independentexperiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005) (E) MDA-MB-231 cells were transfected with flag-vector or vector encoding KLF17 plasmid for 24 hrand cell migration assay was performed Data are representative of three independent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 0005) (F) MDA-MB-231 cells were transfected with control siRNA or siRNA targeting against KLF17 (20 nM) and cell invasion assay was performed Data are representative of three independentexperiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005)


0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

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08

1

12

Rela

tive

klf1

7 m

RNA

leve

l


-190 GAAACACACAGTCTCACCCAGGTAGTTTAGATT -157CD44

-1048 TAAGTTTACCTTTCCACCCACTTTCTCATTTTTG -1014Cyclin-D1

-583 TGAGGCAGGAGGATCACCCAAGGCTAGGAGTTC -550PAI-1

KLF17 binding sequence

A B C D

E F

G

MDA-MB-1386 MDA-MB-1386 MDA-MB-1386 MDA-MB-1386

Flag-KLF17 - + - + - +Input IgG KLF17

CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386

Fig 8 KLF17 inhibits metastasis by targeting key genes in breast cancer cells (AndashD) MDA-MB-1386 cells were transfected with control siRNA or siRNA targeting againstKLF17 (20 nM) for 48 hr and total RNA was extracted to perform qRT-PCR Data are representative of three independent experiments with mean plusmn sd (Two-tailed Studentrsquost-test P lt 0005) (E) Schematic representation of putative KLF17 binding sequence (KLF17BS) within target gene promoters (FndashH) MDA-MB-1386 cells were transfectedwith flag-vector or vector encoding KLF17 plasmid for 24 hr and ChIP assay was performed with indicated antibodies

KLF17RE

EMT target genespromoters

Z0-1 E-cadherinID-1 Vimentin andFibronectin

Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53

Fig 9 Model depicting mutant p53 gain-of-function via inactivation of KLF17 in invasive breast carcinoma (a) Represents that KLF17 inhibits cancer progression in theabsence of mutant p53 (b) Gain-of-function (GOF) of mutant p53 mutant p53 inactivates KLF17 via proteinndashprotein interaction and decreases the recruitment of KLF17 toEMT-related target gene promoters Thus inactivation of KLF17 by mutant p53 may lead to cancer progression in invasive breast carcinoma


Conflict of interests

The authors declare no competing financial interests

Author contributions

Amjad Ali designed the project performed major experimentsand contributed to writing Abdus Saboor Shah performed experi-ments and analyzed data Ayaz Ahmad designed the project andwrote the manuscript

Acknowledgement

We are thankful to our lab colleagues for helpful technical sug-gestions and critically discussing our data Higher EducationCommission (HEC) and the Ministry of Education (MOE) of Paki-stan supported this study

References

[1] D Walerych M Napoli L Collavin G Del Sal The rebel angel mutant p53 asthe driving oncogene in breast cancer Carcinogenesis 33 (2012) 2007ndash2017

[2] M Olivier M Hollstein P Hainaut TP53 mutations in human cancers originsconsequences and clinical use Cold Spring Harbor Perspect Biol 2 (2010)a001008

[3] G Bossi E Lapi S Strano C Rinaldo G Blandino A Sacchi Mutant p53 gainof function reduction of tumor malignancy of human cancer cell lines throughabrogation of mutant p53 expression Oncogene 25 (2006) 304ndash309

[4] LY Lim N Vidnovic LW Ellisen CO Leong Mutant p53 mediates survivalof breast cancer cells Br J Cancer 101 (2009) 1606ndash1612

[5] GA Lang T Iwakuma YA Suh G Liu VA Rao JM Parant et al Gain offunction of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndromeCell 119 (2004) 861ndash872

[6] PA Muller KH Vousden Mutant p53 in cancer new functions and therapeuticopportunities Cancer Cell 25 (2014) 304ndash317

[7] X Lu DP Liu Y Xu The gain of function of p53 cancer mutant in promotingmammary tumorigenesis Oncogene 32 (2013) 2900ndash2906

[8] C Heinlein F Krepulat J Lohler D Speidel W Deppert GV Tolstonog Mutantp53(R270H) gain of function phenotype in a mouse model for oncogene-inducedmammary carcinogenesis Int J Cancer 122 (2008) 1701ndash1709

[9] SI King CA Purdie SE Bray PR Quinlan LB Jordan AM Thompson et alImmunohistochemical detection of Polo-like kinase-1 (PLK1) in primary breastcancer is associated with TP53 mutation and poor clinical outcome BreastCancer Res 14 (2012) R40

[10] DP Liu H Song Y Xu A common gain of function of p53 cancer mutants ininducing genetic instability Oncogene 29 (2010) 949ndash956

[11] W Yan X Chen Identification of GRO1 as a critical determinant for mutantp53 gain of function J Biol Chem 284 (2009) 12178ndash12187

[12] S Di Agostino S Strano V Emiliozzi V Zerbini M Mottolese A Sacchi et alGain of function of mutant p53 the mutant p53NF-Y protein complex revealsan aberrant transcriptional mechanism of cell cycle regulation Cancer Cell 10(2006) 191ndash202

[13] WA Freed-Pastor H Mizuno X Zhao A Langerod SH Moon RRodriguez-Barrueco et al Mutant p53 disrupts mammary tissue architecturevia the mevalonate pathway Cell 148 (2012) 244ndash258

[14] P Stambolsky Y Tabach G Fontemaggi L Weisz R Maor-Aloni Z Siegfriedet al Modulation of the vitamin D3 response by cancer-associated mutant p53Cancer Cell 17 (2010) 273ndash285

[15] W Yan S Liu E Xu J Zhang Y Zhang X Chen et al Histone deacetylaseinhibitors suppress mutant p53 transcription via histone deacetylase 8Oncogene 32 (2013) 599ndash609

[16] G Fontemaggi S DellrsquoOrso P Muti G Blandino S Strano Id2 gene is atranscriptional target of the protein complex mutant p53E2F1 Cell Cycle 9(2010) 2464ndash2466

[17] M Olivier A Langerod P Carrieri J Bergh S Klaar J Eyfjord et al The clinicalvalue of somatic TP53 gene mutations in 1794 patients with breast cancer ClinCancer Res 12 (2006) 1157ndash1167

[18] K Gumireddy A Li PA Gimotty AJ Klein-Szanto LC Showe D Katsaros et alKLF17 is a negative regulator of epithelial-mesenchymal transition andmetastasis in breast cancer Nat Cell Biol 11 (2009) 1297ndash1304

[19] Z Sun Q Han N Zhou S Wang S Lu C Bai et al MicroRNA-9 enhancesmigration and invasion through KLF17 in hepatocellular carcinoma Mol Oncol7 (2013) 884ndash894

[20] IA Ismail HS Kang HJ Lee JK Kim SH Hong DJ-1 upregulates breast cancercell invasion by repressing KLF17 expression Br J Cancer 110 (2014) 1298ndash1306

[21] IA Ismail HS Kang HJ Lee H Chang J Yun CW Lee et al2-Hydroxycinnamaldehyde inhibits the epithelial-mesenchymal transition inbreast cancer cells Breast Cancer Res Treat 137 (2013) 697ndash708

[22] MP Iwanicki JS Brugge Transcriptional regulation of metastatic [Id]entity byKLF17 Genome Biol 10 (2009) 244

[23] FY Liu YL Deng Y Li D Zeng ZZ Zhou DA Tian et al Down-regulated KLF17expression is associated with tumor invasion and poor prognosis inhepatocellular carcinoma Med Oncol 30 (2013) 425

[24] XD Cai YB Zhou LX Huang QL Zeng LJ Zhang QQ Wang et al Reducedexpression of Kruppel-like factor 17 is related to tumor growth and poorprognosis in lung adenocarcinoma Biochem Biophys Res Commun 418 (2012)67ndash73

[25] A Ali Z Wang J Fu L Ji J Liu L Li et al Differential regulation of theREGgamma-proteasome pathway by p53TGF-beta signalling and mutant p53in cancer cells Nat Commun 4 (2013) 2667

[26] G Fontemaggi S DellrsquoOrso D Trisciuoglio T Shay E Melucci F Fazi et alThe execution of the transcriptional axis mutant p53 E2F1 and ID4 promotestumor neo-angiogenesis Nat Struct Mol Biol 16 (2009) 1086ndash1093

[27] AE Knowell D Patel DJ Morton P Sharma S Glymph J Chaudhary Id4dependent acetylation restores mutant-p53 transcriptional activity Mol Cancer12 (2013) 161

[28] P Dong M Kaneuchi Y Xiong L Cao M Cai X Liu et al Identification of KLF17as a novel epithelial to mesenchymal transition inducer via direct activationof TWIST1 in endometrioid endometrial cancer Carcinogenesis 35 (2014)760ndash768

[29] J van Vliet LA Crofts KG Quinlan R Czolij AC Perkins M Crossley HumanKLF17 is a new member of the SpKLF family of transcription factors Genomics87 (2006) 474ndash482

[30] N Montgomery A Hill S McFarlane J Neisen A OrsquoGrady S Conlon et alCD44 enhances invasion of basal-like breast cancer cells by upregulating serineprotease and collagen-degrading enzymatic expression and activity BreastCancer Res 14 (2012) R84

[31] Z Yu L Wang C Wang X Ju M Wang K Chen et al Cyclin D1 induction ofDicer governs microRNA processing and expression in breast cancer NatCommun 4 (2013) 2812

[32] MD Sternlicht AM Dunning DH Moore PD Pharoah DG Ginzinger K Chinet al Prognostic value of PAI1 in invasive breast cancer evidence that tumor-specific factors are more important than genetic variation in regulating PAI1expression Cancer Epidemiol Biomarkers Prev 15 (2006) 2107ndash2114

[33] WB Liu X Jiang F Han YH Li HQ Chen Y Liu et al LHX6 acts as a novelpotential tumour suppressor with epigenetic inactivation in lung cancer CellDeath Dis 4 (2013) e882


Gain-of-function of mutant p53 mutant p53 enhances cancer progression by inhibiting KLF17 expression in invasive breast carcinoma cells

Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results



Mutant p53 recruits to KLF17 promoter in metastatic breast carcinoma

Mutant p53 antagonizes KLF17 mediated EMT genes expression in metastatic breast carcinoma

Mutant p53 prevents recruitment of KLF17 to EMT target gene promoters

KLF17 suppresses cancer progression in advance metastatic breast cancer cells


KLF17 represses CD44 Cyclin-D1 and PAI-1 to inhibit metastasis in breast cancer cells

Discussion



Acknowledgement

References

express low level of KLF17 [182122] Moreover knockdown of KLF17enhances metastasis and EMT in vivo [1819] In contrast forcedexpression of KLF17 inhibits proliferation and growth of cancer cells[2324] Decrease expression of KLF17 is an independent indicatorof poor prognosis for patients with lung adenocarcinomaheptocellular and breast carcinoma [181924] Patients with lowerexpression of KLF17 had greater tumor size later pathological stageand poor prognosis [2324] Although lines of evidence suggest thatKLF17 inhibits cancer progression the signaling pathways control-ling KLF17 function largely remains unknown

Here we show that the expression of metastatic suppressor KLF17is controlled by mutant p53 proteins to potentiate cancer progres-sion in metastatic breast carcinoma Mutant p53 exerts its novel gain-of-function (GOF) ability to inhibit KLF17 expression KLF17 lowexpression in metastatic breast cancer cells can be specifically drivenby mutant p53 that is recruited to upstream of KLF17 promoter Fur-thermore endogenous mutant p53 proteins such as p53-R280K andp53-R282W have the ability to form a complex with KLF17 and an-tagonize KLF17 mediated EMT genes transcription Forced expressionof KLF17 decreases drug resistance in response to chemotherapyIntroduction of KLF17 to mutant p53 containing breast cancer cellssignificantly inhibited migration and invasion by repressing anetwork of oncogenes including PAI-1 CD44 and Cyclin-D1 Our

finding demonstrates that repression of KLF17 by mutant p53 hasa key role in enhancing cancer progression



pcDNA31-R175H pcDNA31-R273H pcDNA31-p53 plasmids were described inour previous study [25] H1299 MDA-MB-1386 and MDA-MB-231 cancer cell lineswere transfected with Lipofectamine 2000 following manufacturerrsquos protocol(Invitrogen)

Antibodies

The following antibodies were used in Western Blot and ChIP experiments anti-p53 (DO-1 Santa Cruz) anti-KLF17 (Abcam) and anti-GAPDH (Santa Cruz)


H1299 MDA-MB-1386 MDA-MB231 and MCF-7 cells were described previouslyin our study [25] For cell treatments we used (05 μM) Adriamycin (Sigma-Aldrich)


EMSA were performed as described previously [25] Briefly 2 μg of nuclearextract or different concentration of purified proteins was incubated with

B

C D

0

02

04

06

08

1

12Re

lativ

e m

RNA

leve

l


Mutant p53

0

05

1

15

2

25

3

35

Rela

tive

mRN

A le

vel


KLF17


1023 318

442 1378

CTL-siRNA p53-siRNA

IB p53

IB KLF17

IB GAPDH

WB


A

1123 196

143 112

CTL-siRNA p53-siRNA

IB p53

IB KLF17

IB GAPDH

WB

Non-metastatic breast cancer cellsMCF-7 (p53)

E

1216 398

215 931

IB KLF17

IB p53

IB GAPDH

WB

CTL-siRNA p53-siRNA


Rela

tive

mRN

A le

vel


Mutant p53

Rela

tive

mRN

A le

vel


KLF17


0

02

04

06

08

1

12

0

05

1

15

2

25

3

35

4

Fig 1 Mutant p53 negatively regulates KLF17 in metastatic breast cancer cells (A and B) MDA-MB-231 and MDA-MB-1386 cells were transfected with control siRNA orsiRNA targeting against p53 (20 nM) for 48 hr Total RNA was extracted and subjected to quantitative RT-PCR (qRT-PCR) analysis The average was calculated based on threeindependent experiments with mean plusmn sd (Two-tailed Studentrsquos t-test P lt 005 P lt 0005) (C and D) MDA-MB-231 and MDA-MB-1386 cells were transfected with controlsiRNA or siRNA targeting against p53 (20 nM) for 72 hr and subjected to immunoblot analyses with indicated antibodies (E) MCF-7 cells were transfected with control siRNAor siRNA targeting against p53 (20 nM) for 72 hr and subjected to immunoblot analyses with indicated antibodies









Luciferase assay


RNA interference


RT-PCR


MTT assay


0

5

10

15

20

25

RLU

(-2000+1) (-1566+1) (-965+1)

CTLp53-R175H

Luc

Luc

Luc

+1-2000

+1-1566

+1-965


A B

C

02468

1012141618

p53-R175H 0 10 25 50

RLU

(ng)

RLU


D

0

5

10

15

20

25

30

(ng)




Cell cycle analysis


Cell migration


Cell invasion



Results





KLF1

7 pr

omot

er

-1906-1576

-3424-3121

ChIP


005

115

225

335

4 CTL-siRNA

Mut-p53-siRNA

Fold

rela

tive

to Ig

G

α-IgG α-p53




-1906-1576

-3424-3121

KLF1

7 pr

omot

er

ChIP

A B

C D

E

-1906-1576-3424-3121

+1

ChIP primers

Fold

rela

tive

to Ig

G

α-IgG α-p53


0

1

2

3

4

5

6CTL-siRNA

Mut-p53-siRNA











si-CTL

si-Mut-p53

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

- + - +Flag-KLF17

MDA-MB-231

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

E-cadherin

MDA-MB-231

0

02

04

06

08

1

12

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

Fibronectin

MDA-MB-231

0

02

04

06

08

1

12Re

lativ

e m

RNA

leve

l

- + - +Flag-KLF17

Vimentin

MDA-MB-231

Flag-KLF17- + - +

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

MDA-MB-1386

0

05

1

15

2

25

3

35

4

45

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

E-cadherin

0

02

04

06

08

1

12

14

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Fibronectin

0

02

04

06

08

1

12

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Vimentin si-CTL

si-Mut-p53

A B C D

E F G H








p53 Ab - - +IgG - + -

IP p53

KLF17

MDA-MB-1386 (p53-R282W)

p53 Ab - - +IgG - + -

p53 IP


05

1

15

2

25

3

35

4

45

5

Fold

rela

tive

to Ig

G

ChIP ZO1 promoter


MDA-MB-231

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G

ChIP Vim promoter

α-KLF17 α-KLF17

Flag-KLF17 - + - +

si-CTLsi-Mut-p53

MDA-MB-231

0

05

1

15

2

25

3

35

4

Fold

rela

tive

to Ig

G



ChIP ZO1 promoter

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G


MDA-MB-1386



si-Mut-p53 si-CTL

KLF17-DNA complex

Free probe1 2 3 4 5 6 7

A B C

D E F

Input

Input








Discussion


CTL-siRNA



A B C

D


0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72


0

01

02

03

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05

06


hr 0 24 48 72

OD

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Adriamycin


0

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popt

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0

01

02

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06


OD

valu

e

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hr 0 24 48 72

OD

valu

e

0

01

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A B C

D

0

01

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06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

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06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

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MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

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MDA-MB-231


Mig

ratio

n in

dex

0

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1

0

02

04

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1

12

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0

05

1

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2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References








Luciferase assay


RNA interference


RT-PCR


MTT assay


0

5

10

15

20

25

RLU

(-2000+1) (-1566+1) (-965+1)

CTLp53-R175H

Luc

Luc

Luc

+1-2000

+1-1566

+1-965


A B

C

02468

1012141618

p53-R175H 0 10 25 50

RLU

(ng)

RLU


D

0

5

10

15

20

25

30

(ng)




Cell cycle analysis


Cell migration


Cell invasion



Results





KLF1

7 pr

omot

er

-1906-1576

-3424-3121

ChIP


005

115

225

335

4 CTL-siRNA

Mut-p53-siRNA

Fold

rela

tive

to Ig

G

α-IgG α-p53




-1906-1576

-3424-3121

KLF1

7 pr

omot

er

ChIP

A B

C D

E

-1906-1576-3424-3121

+1

ChIP primers

Fold

rela

tive

to Ig

G

α-IgG α-p53


0

1

2

3

4

5

6CTL-siRNA

Mut-p53-siRNA











si-CTL

si-Mut-p53

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

- + - +Flag-KLF17

MDA-MB-231

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

E-cadherin

MDA-MB-231

0

02

04

06

08

1

12

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

Fibronectin

MDA-MB-231

0

02

04

06

08

1

12Re

lativ

e m

RNA

leve

l

- + - +Flag-KLF17

Vimentin

MDA-MB-231

Flag-KLF17- + - +

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

MDA-MB-1386

0

05

1

15

2

25

3

35

4

45

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

E-cadherin

0

02

04

06

08

1

12

14

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Fibronectin

0

02

04

06

08

1

12

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Vimentin si-CTL

si-Mut-p53

A B C D

E F G H








p53 Ab - - +IgG - + -

IP p53

KLF17

MDA-MB-1386 (p53-R282W)

p53 Ab - - +IgG - + -

p53 IP


05

1

15

2

25

3

35

4

45

5

Fold

rela

tive

to Ig

G

ChIP ZO1 promoter


MDA-MB-231

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G

ChIP Vim promoter

α-KLF17 α-KLF17

Flag-KLF17 - + - +

si-CTLsi-Mut-p53

MDA-MB-231

0

05

1

15

2

25

3

35

4

Fold

rela

tive

to Ig

G



ChIP ZO1 promoter

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G


MDA-MB-1386



si-Mut-p53 si-CTL

KLF17-DNA complex

Free probe1 2 3 4 5 6 7

A B C

D E F

Input

Input








Discussion


CTL-siRNA



A B C

D


0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72


0

01

02

03

04

05

06


hr 0 24 48 72

OD

valu

e

Adriamycin


0

10

20

30

40

50

60

a

popt

osis


0

01

02

03

04

05

06


OD

valu

e

E


hr 0 24 48 72

OD

valu

e

0

01

02

03

04

05











A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

04

06

08

1

12

MDA-MB-231


Mig

ratio

n in

dex

0

01

02

03

04

05

06

07

08

09

1

0

02

04

06

08

1

12

14



0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References


Cell cycle analysis


Cell migration


Cell invasion



Results





KLF1

7 pr

omot

er

-1906-1576

-3424-3121

ChIP


005

115

225

335

4 CTL-siRNA

Mut-p53-siRNA

Fold

rela

tive

to Ig

G

α-IgG α-p53




-1906-1576

-3424-3121

KLF1

7 pr

omot

er

ChIP

A B

C D

E

-1906-1576-3424-3121

+1

ChIP primers

Fold

rela

tive

to Ig

G

α-IgG α-p53


0

1

2

3

4

5

6CTL-siRNA

Mut-p53-siRNA











si-CTL

si-Mut-p53

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

- + - +Flag-KLF17

MDA-MB-231

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

E-cadherin

MDA-MB-231

0

02

04

06

08

1

12

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

Fibronectin

MDA-MB-231

0

02

04

06

08

1

12Re

lativ

e m

RNA

leve

l

- + - +Flag-KLF17

Vimentin

MDA-MB-231

Flag-KLF17- + - +

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

MDA-MB-1386

0

05

1

15

2

25

3

35

4

45

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

E-cadherin

0

02

04

06

08

1

12

14

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Fibronectin

0

02

04

06

08

1

12

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Vimentin si-CTL

si-Mut-p53

A B C D

E F G H








p53 Ab - - +IgG - + -

IP p53

KLF17

MDA-MB-1386 (p53-R282W)

p53 Ab - - +IgG - + -

p53 IP


05

1

15

2

25

3

35

4

45

5

Fold

rela

tive

to Ig

G

ChIP ZO1 promoter


MDA-MB-231

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G

ChIP Vim promoter

α-KLF17 α-KLF17

Flag-KLF17 - + - +

si-CTLsi-Mut-p53

MDA-MB-231

0

05

1

15

2

25

3

35

4

Fold

rela

tive

to Ig

G



ChIP ZO1 promoter

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G


MDA-MB-1386



si-Mut-p53 si-CTL

KLF17-DNA complex

Free probe1 2 3 4 5 6 7

A B C

D E F

Input

Input








Discussion


CTL-siRNA



A B C

D


0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72


0

01

02

03

04

05

06


hr 0 24 48 72

OD

valu

e

Adriamycin


0

10

20

30

40

50

60

a

popt

osis


0

01

02

03

04

05

06


OD

valu

e

E


hr 0 24 48 72

OD

valu

e

0

01

02

03

04

05











A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

04

06

08

1

12

MDA-MB-231


Mig

ratio

n in

dex

0

01

02

03

04

05

06

07

08

09

1

0

02

04

06

08

1

12

14



0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References









si-CTL

si-Mut-p53

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

- + - +Flag-KLF17

MDA-MB-231

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

E-cadherin

MDA-MB-231

0

02

04

06

08

1

12

Rela

tive

mRN

A le

vel

- + - +Flag-KLF17

Fibronectin

MDA-MB-231

0

02

04

06

08

1

12Re

lativ

e m

RNA

leve

l

- + - +Flag-KLF17

Vimentin

MDA-MB-231

Flag-KLF17- + - +

0

05

1

15

2

25

3

35

4

Rela

tive

mRN

A le

vel

ZO-1

MDA-MB-1386

0

05

1

15

2

25

3

35

4

45

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

E-cadherin

0

02

04

06

08

1

12

14

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Fibronectin

0

02

04

06

08

1

12

- + - +Flag-KLF17

MDA-MB-1386

Rela

tive

mRN

A le

vel

Vimentin si-CTL

si-Mut-p53

A B C D

E F G H








p53 Ab - - +IgG - + -

IP p53

KLF17

MDA-MB-1386 (p53-R282W)

p53 Ab - - +IgG - + -

p53 IP


05

1

15

2

25

3

35

4

45

5

Fold

rela

tive

to Ig

G

ChIP ZO1 promoter


MDA-MB-231

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G

ChIP Vim promoter

α-KLF17 α-KLF17

Flag-KLF17 - + - +

si-CTLsi-Mut-p53

MDA-MB-231

0

05

1

15

2

25

3

35

4

Fold

rela

tive

to Ig

G



ChIP ZO1 promoter

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G


MDA-MB-1386



si-Mut-p53 si-CTL

KLF17-DNA complex

Free probe1 2 3 4 5 6 7

A B C

D E F

Input

Input








Discussion


CTL-siRNA



A B C

D


0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72


0

01

02

03

04

05

06


hr 0 24 48 72

OD

valu

e

Adriamycin


0

10

20

30

40

50

60

a

popt

osis


0

01

02

03

04

05

06


OD

valu

e

E


hr 0 24 48 72

OD

valu

e

0

01

02

03

04

05











A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

04

06

08

1

12

MDA-MB-231


Mig

ratio

n in

dex

0

01

02

03

04

05

06

07

08

09

1

0

02

04

06

08

1

12

14



0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References






p53 Ab - - +IgG - + -

IP p53

KLF17

MDA-MB-1386 (p53-R282W)

p53 Ab - - +IgG - + -

p53 IP


05

1

15

2

25

3

35

4

45

5

Fold

rela

tive

to Ig

G

ChIP ZO1 promoter


MDA-MB-231

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G

ChIP Vim promoter

α-KLF17 α-KLF17

Flag-KLF17 - + - +

si-CTLsi-Mut-p53

MDA-MB-231

0

05

1

15

2

25

3

35

4

Fold

rela

tive

to Ig

G



ChIP ZO1 promoter

si-CTLsi-Mut-p53

0

05

1

15

2

25

3

35

4

45

Fold

rela

tive

to Ig

G


MDA-MB-1386



si-Mut-p53 si-CTL

KLF17-DNA complex

Free probe1 2 3 4 5 6 7

A B C

D E F

Input

Input








Discussion


CTL-siRNA



A B C

D


0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72


0

01

02

03

04

05

06


hr 0 24 48 72

OD

valu

e

Adriamycin


0

10

20

30

40

50

60

a

popt

osis


0

01

02

03

04

05

06


OD

valu

e

E


hr 0 24 48 72

OD

valu

e

0

01

02

03

04

05











A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

04

06

08

1

12

MDA-MB-231


Mig

ratio

n in

dex

0

01

02

03

04

05

06

07

08

09

1

0

02

04

06

08

1

12

14



0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References






Discussion


CTL-siRNA



A B C

D


0005

01015

02025

03035

04045

05O

D va

lue

hr 0 24 48 72


0

01

02

03

04

05

06


hr 0 24 48 72

OD

valu

e

Adriamycin


0

10

20

30

40

50

60

a

popt

osis


0

01

02

03

04

05

06


OD

valu

e

E


hr 0 24 48 72

OD

valu

e

0

01

02

03

04

05











A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

04

06

08

1

12

MDA-MB-231


Mig

ratio

n in

dex

0

01

02

03

04

05

06

07

08

09

1

0

02

04

06

08

1

12

14



0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References








A B C

D

0

01

02

03

04

05

06

07

08

09

1

Mig

ratio

n in

dex

MDA-MB-1386


0010203040506070809

1

Mig

ratio

n in

dex

MDA-MB-1386


0

02

04

06

08

1

12

14

16

18

Inva

sion

inde

x

MDA-MB-1386


Mig

ratio

n in

dex


MDA-MB-231

Inva

sion

inde

x


MDA-MB-231FE

0

02

04

06

08

1

12

MDA-MB-231


Mig

ratio

n in

dex

0

01

02

03

04

05

06

07

08

09

1

0

02

04

06

08

1

12

14



0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References

0

05

1

15

2

25

3

Rela

tive

CD44

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

PAI-1

mRN

A le

vel

0

05

1

15

2

25

3

35

4

Rela

tive

Cycl

in-D

1 m

RNA

leve

l

0

02

04

06

08

1

12

Rela

tive

klf1

7 m

RNA

leve

l






A B C D

E F

G



CD44 promoter

KLF17BS

ChIP

MDA-MB-1386


Cyclin-D1 promoter

KLF17BS

ChIP

MDA-MB-1386


PAI-1 promoter

KLF17BS

ChIPH

MDA-MB-1386


KLF17RE



Cancer inhibition

KLF17

KLF17RE



Cancer progression

KLF17 Mutant p53

a) b)

GOFMutant p53







Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References





Acknowledgement


References




































Introduction



Antibodies





Luciferase assay

RNA interference

RT-PCR

MTT assay

Cell cycle analysis

Cell migration

Cell invasion

Results









Discussion



Acknowledgement

References

gain-of-function of mutant p53: mutant p53 enhances cancer progression by inhibiting klf17...

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