Claudin-low Molecular Subtype Research Articles

Claudins as biomarkers of differential diagnosis and prognosis of tumors.

Claudins are a superfamily of transmembrane proteins, the optimal expression and localization of which are important for the normal physiological function of the epithelium and any imbalance may have pathological consequences. Not only insufficient but also excessive production of claudins in cancer cells, as well as their aberrant localization, equally manifest the formation of a malignant phenotype. Many works are distinguished by contradictory data, which demonstrate the action of the same claudins both in the role of tumor-growth suppressors and promoters in the same cancers. The most important possible causes of significant discrepancies in the results of the works are a considerable variability of sampling and the absence of a consistent approach both to the assessment of the immune reactivity of claudins and to the differential analysis of their subcellular localization. Combined, these drawbacks hinder the histological assessment of the link between claudins and tumor progression. In particular, ambiguous expression of claudins in breast cancer subtypes, revealed by various authors in immunohistochemical analysis, not only fails to facilitate the identification of the claudin-low molecular subtype but rather complicates these efforts. Research into the role of claudins in carcinogenesis has undoubtedly confirmed the potential value of this class of proteins as significant biomarkers in some cancer types; however, the immunohistochemical approach to the assessment of claudins still has limitations, needs standardization, and, to date, has not reached a diagnostic or a prognostic value.

MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer

BackgroundRecent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood.MethodsWe used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC.ResultsWe propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients.ConclusionWe identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Antitumor Activity of Vanicoside B Isolated from Persicaria dissitiflora by Targeting CDK8 in Triple-Negative Breast Cancer Cells.

A flavonoid glycoside, quercitrin (1), and two phenylpropanoyl sucrose derivatives, vanicoside B (2) and lapathoside C (3), were isolated for the first time from the herb Persicaria dissitiflora. Vanicoside B (2) exhibited antiproliferative activity against a panel of cancer cell lines in triple-negative breast cancer (TNBC) MDA-MB-231 cells. The underlying mechanisms of the antitumor activity of 2 were investigated in TNBC cells. Upregulation of cyclin-dependent kinase 8 (CDK8) was observed in a claudin-low molecular subtype of TNBC cells. A molecular modeling study indicated that 2 showed a high affinity for CDK8. Further investigations revealed that 2 suppressed CDK8-mediated signaling pathways and the expression of epithelial-mesenchymal transition proteins and induced cell cycle arrest and apoptosis in MDA-MB-231 and HCC38 TNBC cells. Moreover, 2 inhibited tumor growth without overt toxicity in a nude mouse xenograft model implanted with MDA-MB-231 cells. Taken together, these findings demonstrate the significance of CDK8 activity in TNBC and suggest a potential use of 2 as a therapeutic candidate for the treatment of aggressive human triple-negative breast cancer.

Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells.

CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca2+ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca2+ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.

Molecular and Clinical Characterization of a Claudin-Low Subtype of Gastric Cancer.

Claudin-low molecular subtypes have been identified in breast and bladder cancers and are characterized by low expression of claudins, enrichment for epithelial-to-mesenchymal transition (EMT), and tumor-initiating cell (TIC) features. We evaluated whether the claudin-low subtype also exists in gastric cancer. Four hundred fifteen tumors from The Cancer Genome Atlas (TCGA) gastric cancer mRNA data set were clustered on the claudin, EMT, and TIC gene sets to identify claudin-low tumors. We derived a 24-gene predictor that classifies gastric cancer into claudin-low and non-claudin-low subtypes. This predictor was validated with the Asian Cancer Research Group (ACRG) data set. We characterized molecular and clinical features of claudin-low tumors. We identified 46 tumors that had consensus enrichment for claudin-low features in TCGA data set. Claudin-low tumors were most commonly diffuse histologic type (82%) and originally classified as TCGA genomically stable (GS) subtype (78%). Compared with GS subtype, claudin-low subtype had significant activation in Rho family of GTPases signaling, which appears to play a key role in its EMT and TIC properties. In the ACRG data set, 28 of 300 samples were classified as claudin-low tumors by the 24-gene predictor and were phenotypically similar to the initially derived claudin-low tumors. Clinically, claudin-low subtype had the worst overall survival. Of note, the hazard ratios that compared claudin-low versus GS subtype were 2.10 (95% CI, 1.07 to 4.11) in TCGA and 2.32 (95% CI, 1.18 to 4.55) in the ACRG cohorts, with adjustment for age and pathologic stage. We identified a gastric claudin-low subtype that carries a poor prognosis likely related to therapeutic resistance as a result of its EMT and TIC phenotypes.

Abstract 1117: Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets

Abstract Triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. The claudin-low molecular subtype has higher rates of metastases and recurrence. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes. Citation Format: Margarite D. Matossian, Hope E. Burks, Annie C. Bowles, Rachel A. Sabol, Van T. Hoang, Bruce Bunnell, William J. Zuercher, David H. Drewry, Carrow Wells, Krzysztof Moroz, Matthew E. Burow, Bridgette Collins-Burow. Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1117. doi:10.1158/1538-7445.AM2017-1117

Abstract 5120: The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed

Abstract Rationale: High-grade urothelial carcinoma of the bladder is a heterogeneous disease, with molecular subtypes characterized by distinct tumor biologies and prognoses. Our group and others have described the basal and luminal subtypes, and we now report on the discovery of the claudin-low subtype, all of which resemble analogous subtypes in breast cancer. Methods: We analyzed mRNA sequencing and whole exome sequencing data for The Cancer Genome Atlas (TCGA) bladder tumors and tumors collected at UNC. We performed unsupervised hierarchical clustering on relative gene expression values with significance testing using SigClust to identify the claudin-low subtype. Basal, luminal, and claudin-low bladder tumors were compared for enrichment of genetic features (single nucleotide and copy number variation), gene set expression, immune gene signature expression, T cell receptor (TCR) and B cell receptor (BCR) gene segment expression, and number of predicted MHC Class I and Class II neoantigens. We further report on the first use of our VDJician software to reconstruct full-length rearranged BCR sequences from short-read RNA sequencing data in bladder cancer. Results: Claudin-low bladder tumors were defined by low expression of tight junction claudin-proteins, high expression of epithelial-to-mesenchymal transition genes and immune gene signatures, and were associated with reduced overall survival compared to luminal tumors. A minimal gene set classifier to identify claudin-low tumors showed some but not complete overlap with an optimal classifier derived in breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features: increased rates of RB1, EP300, and NCOR1 mutations, increased frequency of EGFR amplification, decreased rates of FGFR3, ELF3, and KDM6A mutations, and decreased frequency of PPARG amplification. Claudin-low tumors showed the highest expression of immune gene signatures (including an immunosuppression signature), however in contrast to basal tumors, increased immune gene signature expression was not associated with prolonged survival. Claudin-low tumors also showed the highest overall expression of rearranged BCR sequences but lowest BCR repertoire diversity. Predicted neoantigen burden did not vary significantly by subtype, however broad cytokine and chemokine expression levels were elevated in claudin-low tumors, potentially related to low PPARG activity driving increased NFKB activity. Conclusions: Claudin-low bladder cancer is a novel molecular subtype with distinct molecular and immunologic features and prognostic significance. Given the presence of dense immune infiltrates, BCR repertoire characteristics consistent with an antigen-driven response, and high expression of immunosuppression genes, claudin-low bladder tumors may be primed to respond to immune checkpoint inhibitor therapy. Citation Format: Benjamin G. Vincent, William Kim, Jordan Kardos, Shengjie Chai, Joel Parker, Lisle Mose, Sara Selitsky, Michael Iglesia, Matthew Milowsky. The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5120.

Claudin-low bladder tumors are immune infiltrated and actively immune suppressed.

We report the discovery of a claudin-low molecular subtype of high-grade bladder cancer that shares characteristics with the homonymous subtype of breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features including increased rates of RB1, EP300, and NCOR1 mutations; increased frequency of EGFR amplification; decreased rates of FGFR3, ELF3, and KDM6A mutations; and decreased frequency of PPARG amplification. While claudin-low tumors showed the highest expression of immune gene signatures, they also demonstrated gene expression patterns consistent with those observed in active immunosuppression. This did not appear to be due to differences in predicted neoantigen burden, but rather was associated with broad upregulation of cytokine and chemokine levels from low PPARG activity, allowing unopposed NFKB activity. Taken together, these results define a molecular subtype of bladder cancer with distinct molecular features and an immunologic profile that would, in theory, be primed for immunotherapeutic response.

Abstract 1987: The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype

Abstract The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by low gene expression of cell junction and adhesion proteins, and de-regulation of tight junction proteins is often been implicated in tumorigenesis. Herein, we sought to define the role of tricellular tight junction (tTJ) proteins in breast cancer and cancer cell behavior with a focus on lipolysis-stimulated lipoprotein receptor (LSR). LSR was expressed in epithelium, endothelium, adipose and stromal cells within the healthy breast tissue, as well as in tumor epithelium. LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, and ERα positive tumors and breast cancer cell lines. Consistent with the role of LSR in tTJs, LSR levels were significantly reduced in claudin-low breast cancer cell lines. Functional studies illustrated that that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may direct collective cell migration. Moreover, re-introduction of LSR in claudin-low breast cancer cell lines reestablished a family of TJ expression, thereby reverting claudin-low lines to other molecular subtypes. Overexpression of LSR enhanced proliferation and survival in anchorage independent conditions, suggesting that reestablishment of tight junctions is not sufficient to inhibit aggressive cell behaviors in vitro. Collectively, these data highlight an unanticipated role for the tTJ protein, LSR in directing aggressive breast cancer behavior. Citation Format: Jodie M. Fleming, Denise K. Reaves, Katerina D. Fagan-Solis, Karen Dunphy, Shannon D. Oliver. The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2014-1987

The Role of Lipolysis Stimulated Lipoprotein Receptor in Breast Cancer and Directing Breast Cancer Cell Behavior

The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.

Claudin expression in high-grade invasive ductal carcinoma of the breast: correlation with the molecular subtype

Claudin proteins are a major component of the tight junctions. Dysregulation of claudin protein expression has been described in a number of malignancies. Gene expression profiling has stratified breast cancers into distinct molecular subtypes: luminal, HER2 positive (HER2+), and basal-like. Recently, a novel claudin-low molecular subtype has been described. In this study, we correlated the expression patterns of claudins with the molecular subtypes of breast cancer. On the basis of immunohistochemical expression, 226 grade 3 invasive ductal carcinomas were stratified into 65 luminal (estrogen receptor positive (ER+)), 65 HER2+, 86 basal-like, including 14 metaplastic carcinomas (ER−, HER2−, CK5/6, and/or epidermal growth factor receptor positive), and 10 unclassified. Tissue microarrays were analyzed for the expression of claudins 1, 3, 4, 7, and 8 by immunohistochemistry and scored semiquantitatively. High levels of expression were detected in 17% of all cases for claudin 1, 32% claudin 3, 41% claudin 4, 44% claudin 7, and 40% claudin 8. Luminal cancers exhibited increased claudins 7 and 8; basal-like tumors demonstrated increased expression of claudins 1 and 4. Low expression of all five claudins was detected in 30 of 226 cases (13%) and this group was designated ‘claudin-low'. The majority of the claudin-low subgroup were basal-like cancers (23 of 30, 77%). In contrast, only 1 of 30 (3%) claudin-low tumors was of the luminal phenotype and 6 of 30 cases (20%) were HER2+ (P<0.001). Within the basal-like subgroup, 64% of the metaplastic and 19% of the non-metaplastic tumors were claudin-low. The claudin-low group was strongly associated with disease recurrence (P=0.0093). In conclusion, this study is the first to examine comprehensively the differential expression of claudins 1, 3, 4, 7, and 8 in the molecular subtypes of high-grade breast cancer. Claudin-low subtype is a frequent phenomenon in metaplastic and basal-like breast cancer and appears to be a strong predictor of disease recurrence.

Cancer stem cells markers CD44, CD24 and ALDH1 in breast cancer special histological types

AimsCD44, CD24 and ALDH1 are the most consistently used biomarkers to identify and characterise the breast cancer stem cell (CSC) phenotype. However, most studies performed until now analysed samples of...

Immunohistochemical features of claudin-low intrinsic subtype in metaplastic breast carcinomas

PurposeThe claudin-low molecular subtype of breast cancer includes triple negative invasive carcinomas, with a high frequency of metaplastic and medullary features. The aim of this study was to evaluate the immunohistochemistry expression of claudins in a series of metaplastic breast carcinomas. We also assessed other claudin-low features, such as the cancer stem cell-like and epithelial-to-mesenchymal transition phenotypes. ResultsThe majority of the cases showed weak or negative staining for membrane claudins expression. We found 76.9% (10/13) low expressing cases for claudin-1, 84.6% (11/13) for claudin-3 and claudin-4, and 92.3% (12/13) for claudin-7. Regarding the cancer stem cell marker ALDH1, 30.8% (4/13) showed positive staining. We also showed that the majority of the cases presented a CD44+CD24−/low phenotype, positivity for vimentin and lack of E-cadherin expression. Interestingly, these claudin-low molecular features were specific of the mesenchymal component of metaplastic breast carcinomas, since its frequency was very low in other breast cancer molecular subtypes, as luminal, HER2-overexpressing and non-metaplastic triple negative tumors. ConclusionsThe negative/low expression of claudins and E-cadherin, high levels of vimentin, and the breast cancer stem cell phenotype suggests that metaplastic breast carcinomas have similar features to the ones included in the claudin-low molecular subtype, specially their mesenchymal components.

P3-16-02: Targeting Tumor Initiating Cells with siRNA/Nanotherapy in Triple Negative Breast Cancer.

Abstract Residual human breast tumor cells after conventional therapies are enriched in tumor-initiating cells (TIC) characterized by CD44+/ CD24-/low/lineage- with self-renewal capacities. Our gene expression analyses in those cells and in breast cancer cells propagated as mammospheres (MSs) reveal an epithelial-mesenchymal-transition (EMT) signature (400 genes) mainly found in claudin-low molecular subtype human breast tumors. We performed lentiviral shRNA knockdown of that signature in MS. Critical shRNAs were found to decrease the TIC subpopulation. Among those genes, we found STAT3 (signal transducer and activator of transcription 3), NTN4 (netrin 4), and RPL39L (ribosomal protein L39-like). Here, we used a multiscale siRNA delivery system targeting those three genes in order to clarify the effect of silencing those genes over the self-renewal capacity on MSs with claudin-low features. To this end, duplex small interfering RNA (siRNA) against STAT3, NTN4, RPL39L, and scrambled siRNA as control, were introduced into neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC), and using mesoporous microscale biodegradable silicon particles as carriers. This multistage siRNA delivery system has been reported as a good approach for sustained gene silencing. Claudin-low-like human breast cancer cell lines (SUM159 and BT549) were plated in 24-well ultralow attachment plates with mammary epithelial growth medium (MEGM) (5,000 cells/well). Both cell lines were then treated with 1μg/well/6 wells of silicon particles loaded with DOPC nanoliposomes/siRNA. The primary MSs were allowed to grow for 3 days. MFs were counted by day 3 with a GelCount colony counter (Oxford Optronix, Oxford, UK). Mammosphere-Forming Efficiency (MSFE) was calculated by dividing the number of MSs by the number of seeded cells. In addition, established MSs were serially passaged by dissociation, and single cells were replated on fresh 24-well ultra-low attachment plates to form secondary mammospheres, which were counted after 3 days. One-way ANOVA and Tukey test were performed. A p value less than 0.05 was considered as significant. Our results show that silencing STAT3, NTN4, and RPL39L significantly reduces the MSFE in both primary (1%, 0.9%, and 1.7% respectively) (Figure 1) and secondary MSs (0.49%, 0.51%, and 0.45% respectively) (Figure 2) when compared to the scrambled control (2.4% and 1 % respectively) in BT549 cells. For SUM159 cells, we did not find any change in primary MSs for STAT3 (2.2%) and NTN4 (2.1%), even we found a higher percentage of MSFE in those cells treated with RPL39L siRNA (3.9%) when compared to the scrambled control (2.1%) (Figure 3). Nevertheless, a lesser MSFE were observed in those cells treated with STAT3 (0.9%, not significant), NTN4 (0.8%) and RPL39L (0.78%) in comparison to control (1.1%) (Figure 4). In conclusion, knocking-down of EMT-related genes (STAT3, NTN4, and RPL39L) decreases significantly the self-renewal capacity in mammospheres derived from claudin-low-like human breast tumor cells, being BT549 cells more sensitive than SUM159 cells to that silencing with siRNA loaded in DOPC nanoliposomes into silicon particles as carriers. Acknowledgments: We thank Fundacion Alfonso Martin Escudero for the personal support of Dr. Sergio Granados-Principal. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-02.

A clinically relevant gene signature in triple negative and basal-like breast cancer.

IntroductionCurrent prognostic gene expression profiles for breast cancer mainly reflect proliferation status and are most useful in ER-positive cancers. Triple negative breast cancers (TNBC) are clinically heterogeneous and prognostic markers and biology-based therapies are needed to better treat this disease.MethodsWe assembled Affymetrix gene expression data for 579 TNBC and performed unsupervised analysis to define metagenes that distinguish molecular subsets within TNBC. We used n = 394 cases for discovery and n = 185 cases for validation. Sixteen metagenes emerged that identified basal-like, apocrine and claudin-low molecular subtypes, or reflected various non-neoplastic cell populations, including immune cells, blood, adipocytes, stroma, angiogenesis and inflammation within the cancer. The expressions of these metagenes were correlated with survival and multivariate analysis was performed, including routine clinical and pathological variables.ResultsSeventy-three percent of TNBC displayed basal-like molecular subtype that correlated with high histological grade and younger age. Survival of basal-like TNBC was not different from non basal-like TNBC. High expression of immune cell metagenes was associated with good and high expression of inflammation and angiogenesis-related metagenes were associated with poor prognosis. A ratio of high B-cell and low IL-8 metagenes identified 32% of TNBC with good prognosis (hazard ratio (HR) 0.37, 95% CI 0.22 to 0.61; P < 0.001) and was the only significant predictor in multivariate analysis including routine clinicopathological variables.ConclusionsWe describe a ratio of high B-cell presence and low IL-8 activity as a powerful new prognostic marker for TNBC. Inhibition of the IL-8 pathway also represents an attractive novel therapeutic target for this disease.

Generation of tumor-initiating cells by exogenous delivery of OCT4transcription factor

IntroductionTumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations.MethodsOCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments.ResultsOTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew.ConclusionsTransduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.

TGFβ/TNFα-Mediated Epithelial–Mesenchymal Transition Generates Breast Cancer Stem Cells with a Claudin-Low Phenotype

Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial-mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer.

Abstract 3341: TGFβ/ TNFα-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype

Abstract Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2011-3341