TP53 Mutation, Epithelial-Mesenchymal Transition, and Stemlike Features in Breast Cancer Subtypes

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1 Journal of Biomedicine and Biotechnology Volume 0, Article ID 0, pages doi:0./0/0 Research Article TP Mutation, Epithelial-Mesenchymal Transition, and Stemlike Features in Breast Cancer Subtypes Danila Coradini, Marco Fornili, Federico Ambrogi, Patrizia Boracchi, and Elia Biganzoli Department of Clinical and Community Health Sciences, Medical Statistics, Biometry and Bioinformatics, University of Milan, Via Vanzetti, 0 Milan, Italy Correspondence should be addressed to Danila Coradini, danila.coradini@yahoo.it Received March 0; Revised May 0; Accepted June 0 Academic Editor: F. C. Schmitt Copyright 0 Danila Coradini et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Altered p protein is prevalently associated with the pathologic class of triple-negative breast cancers and loss of p function has recently been linked to the induction of an epithelial-mesenchymal transition (EMT) and acquisition of stemness properties. We explored the association between TP mutational status and expression of some genes involved in the canonical TGF-β signaling pathway (the most potent EMT inducer) and in two early EMT associated events: loss of cell polarity and acquisition of stemness-associated features. We used a publicly accessible microarray dataset consisting of p-sequenced primary breast cancers. Statistical analysis indicated that mutant p tumors (especially those harboring a severe mutation) were consistent with the aggressive class of triple-negative cancers and that,differently from cell cultures, surgical tumors underexpressed some TGF-β related transcription factors known as involved in EMT (ID, ID, SMAD, SMAD, SMAD, ZEB). These unexpected findings suggest an interesting relationship between p mutation, mammary cell dedifferentiation, and the concomitant acquisition of stemlike properties (as indicated by the overexpression of PROM and NOTCH genes), which improve tumor cells aggressiveness as indicated by the overexpression of genes associated with cell proliferation (CDK, CDK, MKI) and migration (CXCR, MMP).. Introduction TP tumor suppressor is the most commonly altered gene in human breast cancer where it is mutated in about 0 0% []. TP gene mutations result in altered and stable p proteins that function as dominant negative with gain-of-function properties, including drug resistance, and contribute to malignant progression with detrimental effects on patient s outcome []. In particular, clinical evidence indicated that altered p proteins are prevalently associated with the pathologic class of triple-negative breast cancers, that is, tumors characterized by the immunohistochemical expression of basal cytokeratins and epidermal growth factor receptor (EGFR), but negative for estrogen (ER), progesterone receptor (PR), and HER expression [ ]. Recently, triple-negative tumors have been also associated with a new less common subtype, known as claudin-low []. Wild-type p functions as a sequence-specific DNA binding transcription factor that regulates a plethora of target genes involved in DNA repair, cell cycle control, apoptosis, senescence, angiogenesis, and other fundamental biological process []. Therefore, it is not surprising that mutations of TP gene or inactivations of its signaling pathway are prerequisite for the development of tumors. Most mutations occur within the central DNA binding domain (exons ) and, in particular, at several specific amino acids required for DNA binding. According to the type of mutation (point mutation, deletion, insertion, or stop codon), p protein synthesis may be totally inhibited or may generate functionally altered molecules that affect cell homeostasis in a different manner []. In fact, it is well known that not all p mutations have equal effects: some of them confer loss of function, others have a dominant negative effect and still others are classified as wild-type-like

2 Journal of Biomedicine and Biotechnology protein and represent mutant forms with a limited biological effect [9, 0]. Recently, some excellent papers have provided experimental evidence linking loss of p function to the induction of epithelial-mesenchymal transition (EMT) and acquisition of stemness properties in different tumor cell lines [ ]. EMT is a key program in embryonic development, the aberrant reactivation of which may induce progression, invasion, dissemination, and finally metastasis in cancer cells. The most evident peculiarities of a cell undergoing EMT process are loss of the epithelial phenotype and acquisition of mesenchymal features and abnormal motility capabilities [, ]. The most potent inducer of EMT is transforming growth factor-β (TGF-β) that triggers the activity of several transcription factors (ZEB/TCF, ZEB/SIP, Snail, Slug, Twist, and Ids), which in turn repress the expression of genes coding for epithelial markers and activate the expression of mesenchymal genes []. According to recent acquisition, p should prevent EMT by repressing ZEB and ZEB expressions via mirnas activity. Consequently, p lossof-function should downregulate mirnas expression, the transactivation of transcription factors promoting EMT, and the emergence of tumor cells with stemlike properties [ 9]. So far, despite the availability of a huge amount of information on the transcript profile from microarray analysis, the interrelations among p mutations and genes involved in EMT have not been specifically assessed. Therefore, to investigate the association between TP mutational status and EMT process, we interrogated a publicly accessible microarray dataset consisting of p sequenced primary breast cancers [0]. Adopting an unconventional approach, we did not use the whole transcript profile but we selected a priori panel of genes experimentally recognized as involved in the canonical TGF-β signaling pathway and in two early events associated with EMT: loss of epithelial cell polarity and acquisition of stemness-associated features. To delineate a more comprehensive picture of the relationship among p mutation, EMT, and tumor aggressiveness, we also considered some genes involved in cell proliferation, apoptosis, and metastatic spread.. Materials and Methods.. Materials. As reported in the original paper [0], gene expression profile was determined by using the Affymetrix Human Genome HG-UA and -B GeneChip, and microarray dataset while was available at the Array- Express website ( with the accession number E-GEOD-9. Patients and tumors characteristics were provided as supporting information in the original paper [0]... Gene Selection. According to the aim of the study, we selected genes (Table ). Specifically, the panel was composed of genes recognized as involved in TGFβ-induced EMT, involved in epithelial cell plasticity, coding for stemlike properties and involved in cell proliferation, apoptosis, and metastatic spread. In addition, to describe breast cancer subtypes, 9 genes coding for luminal and basal markers were also considered. The genes corresponded to Affymetrix probe sets, as verified by GeneAnnot system v.0 ( geneannot/), that additionally provided information about the quality of each probe set in terms of sensitivity and specificity score [] (Supplementary Table available at doi:0./0/0)... Statistical Analysis. As some genes are recognized by more than a single probe set, each of which characterized by an individual specificity and sensitivity that differently contribute to gene expression value, a gene expression mean value was calculated after weighting each probe set for its own sensitivity and specificity score. Specifically, each expression value (already log transformed in the original dataset) was multiplied for the semisum of sensitivity and specificity scores of the corresponding probe set. Prediction Analysis for Microarray (PAM) analysis was used to identify genes associated with the TP mutation status. PAM methodology minimizes the classification error using cross validation. For the selected genes, shrunken centroids across the different mutation groups were plotted. The FDR level was estimated through a permutation method. To identify the tumors characterized by a similar intrinsic phenotype (tumor subtypes), unsupervised hierarchical cluster analysis was performed using the subset of genes coding for luminal and basal markers, HER-, and claudins. The choice of the number of clusters to be used was supported by mean silhouette values []. PAM methodology was used to detail differential gene expression among clusters []. Principal Components Analysis (PCA) and PCA-based biplots were used to assess gene expression among clusters []. Moreover, for evaluating the associations among genes, specific subsets, not used to build PCA, were passively projected over the PCA-based biplots of intrinsic phenotypes. Allanalyseswereperformedusingopensourcesoftware R.. packages stats, cluster, and HDMD ( Results and Discussion As described in the original paper [0], the cases series was composed of tumors, of which characterized by a TP mutation. Of the mutant tumors, had point mutations and had severe mutations, that is, insertions (n = ), deletions (n = ), and stop codons (n = ), that result in frame shift and truncations with deleterious functional consequences. To identify genes differentially expressed between mutant and wild-type p tumors, we first applied a PAM analysis on the overall cases series (Figure ). According to the expected loss-of-function, mutant p tumors were characterized by the underexpression of genes under p control (i.e., CDKNA, coding for p, and TPINP, coding for the p- inducible nuclear protein ). In addition, they were showed as underxpressed several genes coding for epithelial cell

3 Journal of Biomedicine and Biotechnology Table : Genes entered in the study. Apical junctional complex Apoptosis Basal markers Official gene symbol Gene name Entrez gene ID Ensembl genomic location CDH Cadherin, type, E-cadherin (epithelial) 999 q. CDH Cadherin, type, N-cadherin (neuronal) 000 q. CLDN Claudin 90 q CLDN Claudin 90 Xq. CLDN Claudin q. CLDN Claudin q. CLDN Claudin q. CLDN Claudin 90 p. CLDN Claudin p. CLDN Claudin 90 q. CLDN9 Claudin p. CLDN0 Claudin 0 90 q. CLDN Claudin 00 q. CLDN Claudin 909 q. CLDN Claudin q. CLDN Claudin q. CLDN Claudin 0 q CLDN Claudin q. CLDN Claudin 0 q. CLDN Claudin 0 p. CTNNA Catenin (cadherin-associated protein), alpha 9 q. CTNNB Catenin (cadherin-associated protein), beta 99 p. CTNND Catenin (cadherin-associated protein), delta 00 q. JAM Junctional adhesion molecule 9 q. JAM Junctional adhesion molecule 00 q JUP Junction plakoglobin q. MAGI Membrane associated guanylate kinase, WW and PDZ domain containing 9 p. MARVELD MARVEL domain containing q. OCLN Occludin 90 q. PVRL Poliovirus receptor-related (herpesvirus entry mediator C) q. PVRL Poliovirus receptor-related (herpesvirus entry mediator B) 9 9q. PVRL Poliovirus receptor-related 9 q. PVRL Poliovirus receptor-related 0 q. SYMPK Symplekin 9 9q. TJP Tight junction protein (zona occludens ) 0 q. TJP Tight junction protein (zona occludens ) 9 9q. TJP Tight junction protein (zona occludens ) 9p. VCL Vinculin 0q. BAX BCL-associated X protein 9q. BCL B-cell CLL/lymphoma 9 q. CASP Caspase, apoptosis-related cysteine peptidase q ERBB V-erb-b erythroblastic leukemia viral oncogene homolog (avian) 0 q MDM Mdm p binding protein homolog (mouse) 9 q TP Tumor protein p p. TPINP Tumor protein p inducible nuclear protein 9 q. XIAP X-linked inhibitor of apoptosis Xq ACTA Actin, alpha, skeletal muscle q. CD CD molecule (Indian blood group) 90 p EGFR Epidermal growth factor receptor 9 p. KRT Keratin q. KRTA Keratin A q. KRTB Keratin B q.

4 Journal of Biomedicine and Biotechnology Cell cycle Cell polarity complexes Epithelialmesenchymal transition Table : Continued. Official gene symbol Gene name Entrez gene ID Ensembl genomic location KRT Keratin q. KRT Keratin q. TP Tumor protein p q VIM Vimentin 0p CCNB Cyclin B Xp. CCND Cyclin D 9 q. CCNE Cyclin E 9 q. CDK Cyclin-dependent kinase 0 q. CDK Cyclin-dependent kinase 09 q. CDK Cyclin-dependent kinase 0 q. CDKNA Cyclin-dependent kinase inhibitor A (p, Cip) 0 p. CDKNB Cyclin-dependent kinase inhibitor B (p, Kip) 0 p. CDKNA Cyclin-dependent kinase inhibitor A (melanoma, p, CDK) 09 9p. CDKNB Cyclin-dependent kinase inhibitor B (p, inhibits CDK) 00 9p. CDKND Cyclin-dependent kinase inhibitor D (p9, inhibits CDK) 0 9p. GAS Growth arrest-specific 9 9q. MKI Antigen identified by monoclonal antibody Ki- 0q. CRB Crumbs homolog (Drosophila) q. CRB Crumbs homolog (Drosophila) 99 9p. DLG Discs, large homolog (Drosophila) 9 q9 DLG Discs, large homolog (Drosophila) 0 q. DLG Discs, large homolog (Drosophila) Xq. DLG Discs, large homolog (Drosophila) p. DLG Discs, large homolog (Drosophila) 9 0q. INADL InaD-like (Drosophila) 00 p. MPP Membrane protein, palmitoylated (MAGUK p subfamily member ) 9 q. LLGL Lethal giant larvae homolog (Drosophila) 99 p. LLGL Lethal giant larvae homolog (Drosophila) 99 q. PARD Par- partitioning defective homolog (C. elegans) 0p. PARDB Par- partitioning defective homolog B (C. elegans) q. PARDA Par- partitioning defective homolog alpha (C. elegans) 0 q. PARDB Par- partitioning defective homolog beta (C. elegans) 0q. PARDG Par- partitioning defective homolog gamma (C. elegans) q PRKCI Protein kinase C, iota q. PRKCZ Protein kinase C, zeta 90 p. SCRIB Scribbled homolog (Drosophila) q. AKT V-akt murine thymoma viral oncogene homolog 0 q. AKT V-akt murine thymoma viral oncogene homolog 0 9q. BMP Bone morphogenetic protein 9 p. ID Inhibitor of DNA binding, dominant negative helixloop-helix protein 9 0q. ID Inhibitor of DNA binding, dominant negative helixloop-helix protein 9 p. ID Inhibitor of DNA binding, dominant negative helixloop-helix protein 99 p. ID Inhibitor of DNA binding, dominant negative helixloop-helix protein 00 p. SMAD SMAD family member 0 q. SMAD SMAD family member 0 q. SMAD SMAD family member 09 q.

5 Journal of Biomedicine and Biotechnology Luminal markers Metastasis-related genes Stemlike features Table : Continued. Official gene symbol Gene name Entrez gene ID Ensembl genomic location SMAD SMAD family member 090 q. SMAD SMAD family member 09 q. SMAD SMAD family member 09 q. SMURF SMAD specific E ubiquitin protein ligase q. SMURF SMAD specific E ubiquitin protein ligase 0 q. SNAI Snail homolog (Drosophila) 0q. SNAI Snail homolog (Drosophila) 9 q. TCF Transcription factor (EA immunoglobulin enhancer binding factors E/E) 99 9p. TGFBI Transforming growth factor, beta-induced, kda 0 q. TGFBR Transforming growth factor, beta receptor 0 9q. TGFBR Transforming growth factor, beta receptor II 0 p. TGFBR Transforming growth factor, beta receptor III 09 p. TGIF TGFB-induced factor homeobox 0 0q. TWIST Twist homolog (Drosophila) 9 p. TWIST Twist homolog (Drosophila) q. ZEB Zinc finger E-box binding homeobox 9 0p. ZEB Zinc finger E-box binding homeobox 99 q. CD CD molecule 009 q ESR Estrogen receptor 099 q. GATA GATA binding protein 0p KRT Keratin q. KRT Keratin q. KRT Keratin q. KRT9 Keratin 9 0 q. MUC Mucin, cell surface associated q PGR Progesterone receptor q. CXCR Chemokine (C-X-C motif) receptor q. CXCR Chemokine (C-X-C motif) receptor q. MMP Matrix metallopeptidase (interstitial collagenase) q. MMP Matrix metallopeptidase (gelatinase A, kda gelatinase, kda type IV collagenase) q. MMP Matrix metallopeptidase (stromelysin, progelatinase) q. MTA Metastasis associated 9 q. MTA Metastasis associated family, member 99 q. MTA Metastasis associated family, member 0 p PAK P protein (Cdc/Rac)-activated kinase 9 q. TIMP TIMP metallopeptidase inhibitor 0 Xp. ABCG ATP-binding cassette, sub-family G (WHITE), member 99 q. ALDHA Aldehyde dehydrogenase family, member A 9q. ALDHA Aldehyde dehydrogenase family, member A 0 q. BMI BMI polycomb ring finger oncogene 0p. JAG Jagged 0p. JAG Jagged q. NANOG Nanog homeobox 99 p. NOTCH Notch 9q. NOTCH Notch homolog (Drosophila) p NOTCH Notch homolog (Drosophila) 9p. NOTCH Notch homolog (Drosophila) p. NUMB Numb homolg (Drosophila) 0 q. PROM Prominin p.

6 Journal of Biomedicine and Biotechnology MKI MMP TJP NANOG ZEB JAM CDK TGFBR CXCR MAGI ID PARDB INADL DLG ALDHA PROM CLDN BCL TCF CDK PARDB CCND JAM TJP TPINP PRKCI SMAD CDH PVRL CDKNA BAX CCNE NOTCH CDKNA SMAD MMP CLDN TGIF DLG ABCG SMAD CRB TGFBR CDKND GAS SMURF SYMPK CTNNA MDM TIMP AKT JAG CDKNB OCLN TJP NUMB CLDN TGFBI CTNNB TP CDK PVRL CTNND TP wild-type TP mutant Figure : Shrunken centroids for wild-type TP and mutant TP tumors. Left-sided bars indicate lower expression in the subgroups relative to overall centroid; right-sided bars indicate higher expression in subgroups relative to overall centroid. polarity complex (DLG, DLG, INADL, PARDB, PARDB) and apical junctional components (CLDN, JAM, JAM, MAGI, OCLN, PVRL, TJP, TJP). Conversely, mutant p tumors overexpressed CDH, the gene coding for N- cadherin, and some genes involved in cell proliferation (CCNE, CDK, CDK, CDKNA, CDKND, MKI) and metastatic spread (CXCR, MMP). An overall similar pattern of expression was found when we interrogated another publicly available microarray dataset where p mutational status was known [] using the same panel of genes. Despite the different microarray platform used (K cdna microarrays instead of Affymetrix GeneChip), mutant p tumors were associated with a dramatic underexpression of genes coding for apical junctional components (INADL, JAM, JAM, MAGI, PARDB, PVRL) coupled with the overexpression of genes coding for N-cadherin (CDH) or involved in cell proliferation and metastatic spread (CCNE, CDK, CDK, MKI, MTA) (Supplementary Figure ). Notably, in both datasets, p mutant tumors were associated with the overexpression of PROM, supporting the experimental evidence indicating the relation among p mutation and the reacquisition of some stemlike properties according to an EMT-like process [, ]. As regards the genes related to the canonical TGF-β signaling pathway, mutant p tumors showed the downregulation of many genes coding for pivotal elements of this pathway (ID, ID, SMAD, SMAD, SMAD, TGFBR, TGFBR, ZEB) coupled with the overexpression of SMURF, coding for a SMAD-specific E ubiquitin protein ligase, and TGIF, coding for a transcriptional repressor interacting with TGF-β activated SMAD proteins (Figure ). A similar pattern of expression (i.e., downregulation of SMAD, SMAD, SMAD, SMAD, TGFBR, ZEB, and overexpression of SMURF and TGIF) was found in Langerod dataset [] (Supplementary Figure ). This unexpected finding could be explained taking into account that TGF-β is a multifunctional cytokine and a powerful tumor suppressor that governs many aspects of mammary epithelial cells physiology and homeostasis []. Consistent with the notion that estrogen receptor and TGFβ signaling pathways are major regulators during mammary

7 Journal of Biomedicine and Biotechnology AKT AKT BMP ID ID ID ID All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut SMAD SMAD SMAD SMAD SMAD SMAD SMURF All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut SMURF SNAI SNAI TCF TGFBI TGFBR TGFBR All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut TGFBR TGIF TWIST TWIST ZEB ZEB All WT mut All WT mut All WT mut All WT mut All WT mut All WT mut Figure : Boxplots of the genes associated with EMT in wild-type (WT) or mutant (mut) TP tumors with respect to overall case series (all). gland development [], it is not surprising that p mutant tumors concomitantly underexpressed ESR (coding for ERα), TGFBR, TGFBR (coding for TGF-β receptors) and ID, ID, SMAD, SMAD, SMAD, ZEB (coding for key elements of the pathway). When severe and missense mutations were considered separately, PAM analysis provided evidence that severe TP mutations were responsible for the differential gene expression observed in mutant with respect to wild-type p tumors, even though some important alterations were already present in missense TP mutations, as, for example, the downregulation of some apical junctional components or the overexpression of genes related to cell proliferation and invasion. As shown in Figure, in addition to the expected decrease in the expression of TP, CDKNA, and TPINP, tumors harboring severe mutations were characterized by a dramatic overexpression of genes associated with proliferation (CDK, CDK, MKI) and metastatic spread (CXCR, MMP), and by underexpression of several genes involved in epithelial cell identity (DLG, INADL, JAM, JAM, MAGI, OCLN, PARDB, PARDB, PVRL, SCRIB, TJP and TJP). As regards the association between missense or severe TP mutation and EMT-related genes, Figure indicates that, with respect to tumors harboring a missense mutation, those with severe mutations were characterized by the overexpression of SMURF, SNAI, and TGIF genes. Of particular interest is the overexpression of SNAI gene because of the concomitant overexpression of NOTCH pointed out by PAM analysis in tumors with severe TP mutation (Figure ). Indeed, Notch signalling pathway, which is implicated as an important contributor to EMT in tumorigenesis, has been recently suggested to play a direct role on the expression of the Snail transcription factor []. With respect to missense TP mutations, severe ones were also characterized by an increased expression of PROM, the gene encoding for prominin, a pentaspan transmembrane glycoprotein (CD) often overexpressed on cancer cells, where it is thought to function in maintaining stem cell properties by suppressing differentiation. This finding is in agreement with the hypothesis that basal cancers, which have been proposed to have a stem cell origin, are virtually all TP mutantsandexpresshighlevelsof PROM transcript and protein []. Unfortunately, since in Langerod dataset [] severe mutations accounted for only three cases, we were unable to verify all these observations in an independent dataset.

8 Journal of Biomedicine and Biotechnology TJP CDK MKI TP MMP TPINP JAM PROM PARDB PVRL CLDN NOTCH ALDHA PRKCI DLG CTNNA ID ZEB TJP NANOG CDH ALDHA JAM CXCR PARDB TGFBR MAGI TGIF SMURF SNAI CDKNA INADL CCND TCF ID CDK CDKNA CLDN OCLN BCL ID AKT SMAD ABCG SCRIB CLDN PVRL CLDN MMP SMAD GAS TIMP AKT CLDN CLDN CDK CTNND SMAD MDM DLG PAK NUMB BMI BAX TGFBR CLDN CCNE CTNNB DLG TWIST DLG CRB CDKND SYMPK JAG SMAD CDKNB TJP JUP LLGL TGFBI SNAI PVRL ZEB TP wild-type TP mut (missense) TP mut (severe) Figure : Shrunken centroids for wild-type TP an mutant (missense or severe mutation) TP tumors. Left-sided bars indicate lower expression in the subgroups relative to overall centroid; right-sided bars indicate higher expression in subgroups relative to overall centroid. One of the aims of the study was to explore the relationship among p mutation, EMT, and tumor aggressiveness, a peculiar characteristic of certain breast cancer subtypes (especially basal-like phenotype). To this specific aim, we performed an unsupervised hierarchical cluster analysis, using the subset of genes coding for luminal and basal markers, HER-, and claudins, and we looked at the distribution of p mutations according to tumor subtype. The analysis indicated that mutant p tumors distributed into three main clusters (Figure ). Of the mutant p tumors, were included in Cluster, in Cluster, and in Cluster. However, looking at the relative percentage, we found that, on the total number of tumors in each cluster, only % (/) of Cluster and 9% (/9) of Cluster tumors had p mutations, whereas % (/) of Cluster tumors did have. Notably, 0 of these mutations were severe mutations. PCA-based biplots, drawn using the same subset of genes of hierarchical cluster analysis (Figure ), showed that Cluster tumors were positively associated with genes related to basal phenotype (KRT, KRTA, KRTB, KRT, KRT, EGFR) and with a panel of claudin-coding genes (CLDN, CLDN, CLDN0), whereas they were negatively associated with the majority of genes related to luminal phenotype. Conversely, Cluster tumors were positively associated with genes related to luminal phenotype (ESR, GATA, MUC, PGR, KRT) and with a different panel of claudin-coding genes (CLDN, CLDN, CLDN) and negatively associated with genes related to basal phenotype. Cluster tumors showed a less clear-cut phenotype according to the more heterogeneous nature of this cluster, even though they appeared prevalently, associated with genes related to basal phenotype (KRT, KRTB, KRT, KRT, TP). Remarkably, Cluster tumors also showed the concomitant underexpression of ERBB gene providing evidence that these tumors had a gene expression profile consistent with the pathologic class of triple-negative tumors (Supplementary Figure ), which are characterized by the expression of basal cytokeratins (mainly Krt) and EGFR, but do not express estrogen and progesterone receptors, and HER. When we looked at the expression of EMT-associated genes according to clusters (Figure ), we found that Cluster, consistent with the pathologic class of triple-negative cancers, showed a gene expression profile similar to that of tumors harboring a severe p mutation. Conversely, Cluster, consistent with the luminal-like phenotype, had a pattern of expression similar to that of wild-type p tumors whereas the phenotypically heterogenous Cluster looks like the group of tumors with a missense p mutation. In particular,

9 Journal of Biomedicine and Biotechnology AKT AKT BMP ID ID ID ID All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV SMAD SMAD SMAD SMAD SMAD SMAD SMURF All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV SMURF SNAI SNAI. TCF TGFBI TGFBR TGFBR All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV TGFBR TGIF TWIST TWIST ZEB ZEB All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV All WTMS SV Figure : Boxplots of the genes associated with EMT in wild-type (WT), missense (MS), or severe (SV) TP mutation with respect to overall case series (all). 0 0 TP Cluster Cluster Cluster Figure : Tumor dendrogram from clustering tumors and 0 genes associated with intrinsic breast cancer phenotypes (luminal, basal, ERBB, and claudins). Black bars denote tumors with a wild-type p; orange bars, tumors with a mutant p (missense mutation); red bars, tumors with a mutant p (severe mutation). Cluster (consistent with the pathologic class of triplenegative cancers and akin to severe p mutated tumors) was characterized by the underexpression of SMAD, SMAD, ZEB, and TGFBR and the overexpression of SMURF, TGIF, and SNAI, in agreement with the gene profile observed in tumors harboring a severe TP mutation (Figure ). Notably, when the subset of genes related to stemness properties was passively projected over the PCA-based biplots provided in Figure, Cluster tumors were positively associated with PROM and NOTCH, and negatively associated with ALDHA, BMI, NUMB (Figure ). Similar to the latter but opposite in the sign, was the pattern of association shown by Cluster tumors.

10 0 Journal of Biomedicine and Biotechnology PC (.%) 0 CLDN KRT CLDN KRT KRT KRT9 CDERBB CLDN ACTA KRTA CLDN9 KRT EGFR KRTB CLDN CLDN0 CLDN CLDN CLDN GATA KRT KRT TP MUC CD PGR ESR VIM CLDN PC (9.%) 0 CLDN CLDN0 CD CLDN KRTA EGFR CLDN9 CLDN ACTA CD ERBB CLDN KRT VIM CLDN CLDN KRTB CLDN KRT KRT KRT9 KRT KRT CLDN ESR KRT TP MUC GATA PGR 0 Basal Luminal Claudins ERBB PC (9.9%) Mutant Wild-type 0 Basal Luminal Claudins ERBB PC (9.9%) Mutant Wild-type (a) (b) Figure : Active PCA-based biplots of intrinsic breast cancer phenotype-related genes. Samples are colored according to the mutational p status (wild-type in green and mutant in blue) and are labeled according to the corresponding cluster (,, or ). Loading for basal genes are represented by red arrows, luminal genes by pink arrows, claudin genes by orange arrows, and ERBB gene by yellow arrow. The imbalance in Numb/Notch pathway observed in Cluster tumors, associated with the overexpression of SNAI, is of particular interest because the involvement of this pathway in differentiation program and epithelial cancer progression and metastasis. Numb is an evolutionary conserved protein that plays a critical role in cellfate determination, including control of asymmetric cell division, endocytosis, cell adhesion, cell migration, and ubiquitination of specific substrates as p. Loss of Numb causes increased activity of the oncogene Notch and for this reason, low expression of Numb and high levels of Notch have been associated with tumor progression and used as markers of tumor aggressiveness, especially in basal-like breast cancer [9]. The aggressiveness of this group of tumors was corroborated by the observation that Cluster tumors were prevalently poorly differentiated (/ tumors were Grade III) with respect to Cluster and Cluster tumors, and by the positive association with genes promoting cell proliferation and metastatic spread. In this context, it can be viewed the overexpression of MMP and CXCR, and the downregulation of TIMP. Indeed, MMP encodes for a matrix metalloproteinase family member (specifically, a collagenase) involved in the breakdown of extracellular matrix whereas TIMP encodes for a specific tissue inhibitor of metalloproteases, including MMP-. Because MMP is a target gene for wild-type p activity, the functional inactivation of the protein results in a gene overexpression that allows tumor cell migration after degradation of basement membrane and cell detachment [0, ]. The concomitant overexpression of CXCR due to a gain-of-function mutant p [, ], further contributes to enhance tumor cell migration and metastatic spread []. In fact, CXCR encodes a C-X-C motif chemokine receptor specific for stromal cell-derived factor- (SDF-/CXCL), a member of the family of chemoattractant molecules, physiologically involved in the migration of immune cells. The CXCL/CXCR signaling axis is also known to be important for tumor cell migration: CXCR expressed on tumor cells, provides a means of homing for metastatic cells to target organs []. Due to its implication with tumor dissemination, CXCR overexpression has been linked to a poor prognosis in breast cancer patients []. Surprisingly, on the contrary, it should be the negative association, pointed out by PCA-based biplots, between Cluster tumors and BMI expression. That, because the role of BMI gene in self-renewal of stem cells and as an oncogene in many human cancers where it induces EMT. Although Bmi overexpression has been correlated with poor prognosis in several tumor types, a recent study has indicated that, in breast cancer, high Bmi expression is limited to the luminal subtype and that it is associated with a good outcome []. Under this light, the positive association that we observed between BMI expression and Cluster tumors, consistent with the luminal-like phenotype, seems to provide a transcriptomic support to this clinical evidence.

11 Journal of Biomedicine and Biotechnology AKT AKT BMP ID ID ID ID All All All All All All All SMAD SMAD SMAD SMAD SMAD SMAD SMURF All All All All All All All SMURF SNAI SNAI TCF TGFBI TGFBR TGFBR All All All All All All All TGFBR TGIF TWIST TWIST ZEB ZEB All All All All All All Figure : Boxplots of the genes associated with EMT in the three main clusters identified by unsupervised hierarchical cluster analysis using the subset of genes coding for luminal and basal markers, HER- and claudins. Cluster tumors, which are prevalently p wildtype, are more difficult to categorize. Dissimilarly from basal-like and luminal-like, these tumors had an indefinite phenotype characterized by the coexpression of luminal and basal cytokeratins. In addition, the overexpression of several transcription factors (ID, ID, SNAI, TWIST, ZEB), known to be under TGF-β control, and the concomitant overexpression of some genes coding for stemlike properties (ABCG, JAG, JAG, NANOG, NOTCH) makes it difficult to have a correct interpretation of the results. Indeed, it is not easy to establish whether such a phenotypical heterogeneity represents an intermediate step of an EMT-like process, in which tumor cells gain characteristics of mesenchymal cells but have not completely lost epithelial characteristics, or it is simply due to the individual heterogeneity of the tumors forming the cluster.. Conclusions Aim of this in silico study was to investigate the association between TP mutational status and expression of a panel of genes related to TGF-β induced EMT and stemlike features, using a publicly accessible microarray dataset consisting of p-sequenced primary breast cancers. According to recent experimental evidence linking loss of p function, induction of EMT and acquisition of stemness properties in different tumor cell lines [ ], we expected an evident positive association between EMT-related genes and p mutations, in particular with severe p mutations. In addition, since clinical evidence indicates that p mutations are prevalently associated with the pathologic class of triple-negative breast cancers, we expected an overexpression of EMT-related genes in this specific subset of tumors. Our analysis supports the notion that mutant p tumors (especially those harboring a severe p mutation) were consistent with the aggressive clinic class of triple-negative cancers, but it clearly indicates that, differently from cell cultures [ ], surgical tumors did not overexpress TGF-β-related transcription factors. Taking into account the physiological role of TGF-β in mammary gland differentiation [, ], these unexpected findings seem to suggest an interesting relationship between p mutation, mammary cell dedifferentiation, and the concomitant acquisition of stemlike properties which improve tumor cells aggressiveness.

12 Journal of Biomedicine and Biotechnology PC (.%) 0 CLDN KRT CLDN KRT KRT KRT9 CD ERBB CLDN ACTA KRTA CLDN9 PROM EGFR KRT CLDN KRTB CLDN0 ALDHA CLDN NOTCH CLDN NOTCH CLDN MUC GATA KRT KRT TP JAG NANOG NUMB BMI NOTCH NOTCH CD ESR JAG PGR VIM CLDN ABCG ALDHA PC (9.%) 0 CLDN CLDN0 NOTCH CD CLDN KRTA PROM EGFR CLDN9 CLDN CD ERBB ACTA NOTCH CLDN KRT ALDHA VIM NOTCH CLDN BMI NOTCH ALDHA NUMB CLDN KRTB CLDNJAG ABCG JAG KRT KRT9 NANOG KRT KRT KRT KRT TP CLDN MUC ESR GATA PGR 0 Basal Luminal Claudins ERBB PC (9.9%) Mutant Wild-type 0 Basal Luminal Claudins ERBB PC (9.9%) Mutant Wild-type (a) (b) Figure : Passive projectionof stemlike-related genes in the space of the first two principal components (PC), (a), and in the space of first and third PCs, (b), of intrinsic phenotype gene set. Loading for EMT or stemlike genes are represented in black. References [] M. Olivier, A. Langerød, P. Carrieri et al., The clinical value of somatic TP gene mutations in,9 patients with breast cancer, Clinical Cancer Research, vol., no., pp., 00. [] A. Di Leo, M. Tanner, C. Desmedt et al., p- gene mutations as a predictive marker in a population of advanced breast cancer patients randomly treated with doxorubicin or docetaxel in the context of a phase III clinical trial, Annals of Oncology, vol., no., pp , 00. [] A. Langerød, H. Zhao, Ø. Borgan et al., TP mutation status and gene expression profiles are powerful prognostic markers of breast cancer, Breast Cancer Research, vol. 9, no., article R0, 00. [] B. J. Chae, J. S. Bae, A. Lee et al., p as a specific prognostic factor in triple-negative breast cancer, Japanese Journal of Clinical Oncology, vol. 9, no., pp., 009. [] E. Biganzoli, D. Coradini, F. Ambrogi et al., p status identifies two subgroups of triple-negative breast cancers with distinct biological features, Japanese Journal of Clinical Oncology, vol., no., pp. 9, 0. [] A. Prat, J. S. Parker, O. Karginova et al., Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer, Breast Cancer Research, vol., no., article R, 00. [] K. H. Vousden and C. Prives, Blinded by the light: the growing complexity of p, Cell, vol., no., pp., 009. [] A. J. Levine and M. Oren, The first 0 years of p: growing ever more complex, Nature Reviews Cancer, vol. 9, no. 0, pp. 9, 009. [9] P. Monti, P. Campomenosi, Y. Ciribilli et al., Tumour p mutations exhibit promoter selective dominance over wild type p, Oncogene, vol., no., pp., 00. [0] J. J. Jordan, A. Inga, K. Conway et al., Altered-function p missense mutations identified in breast cancers can have subtle effects on transactivation, Molecular Cancer Research, vol., no., pp. 0, 00. [] T. Kim, A. Veronese, F. Pichiorri et al., p regulates epithelial-mesenchymal transition through micrornas targeting ZEB and ZEB, Journal of Experimental Medicine, vol. 0, no., pp., 0. [] C.-J. Chang, C.-H. Chao, W. Xia et al., P regulates epithelial-mesenchymal transition and stem cell properties through modulating mirnas, Nature Cell Biology, vol., no., pp., 0. [] A. V. Pinho, I. Rooman, and F. X. Real, p-dependent regulation of growth, epithelial-mesenchymal transition and stemness in normal pancreatic epithelial cells, Cell Cycle, vol. 0, no., pp., 0. [] J. Yang and R. A. Weinberg, Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis, Developmental Cell, vol., no., pp. 9, 00. [] D. S. Micalizzi, S. M. Farabaugh, and H. L. Ford, Epithelialmesenchymal transition in cancer: parallels between normal development and tumor progression, Journal of Mammary Gland Biology and Neoplasia, vol., no., pp., 00. [] J. Zavadil and E. P. Böttinger, TGF-β and epithelial-tomesenchymal transitions, Oncogene, vol., no., pp., 00. [] J. Schubert and T. Brabletz, P spreads out further: suppression of EMT and stemness by activating mir-00c expression, Cell Research, vol., no., pp. 0 0, 0.

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