Primary Human Breast Carcinomas Research Articles

CAR expression in invasive breast carcinoma and its effect on adenovirus transduction efficiency

BackgroundBreast cancer is the second leading cause of death in women, with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) as the two most common forms of invasive breast cancer. While estrogen receptor positive (ER+) IDC and ILC are treated similarly, the multifocality of ILC presents challenges in detection and treatment, worsening long-term clinical outcomes in patients. With increasing documentation of chemoresistance in ILC, additional treatment options are needed. Oncolytic adenoviral therapy may be a promising option, but cancer cells must express the coxsackievirus & adenovirus receptor (CAR) for adenoviral therapy to be effective. The present study aims to evaluate the extent to which CAR expression is observed in ILC in comparison to IDC, and how the levels of CAR expression correlate with adenovirus transduction efficiency. The effect of liposome encapsulation on transduction efficiency is also assessed.MethodsTo characterize CAR expression in invasive breast carcinoma, 36 formalin-fixed paraffin-embedded (FFPE) human breast tumor samples were assayed by CAR immunohistochemistry (IHC). Localization of CAR in comparison to other junctional proteins was performed using a multiplex immunofluorescence panel consisting of CAR, p120-catenin, and E-cadherin. ILC and IDC primary tumors and cell lines were transduced with E1- and E3-deleted adenovirus type 5 inserted with a GFP transgene (Ad-GFP) and DOTAP liposome encapsulated Ad-GFP (DfAd-GFP) at various multiplicities of infection (MOIs). Transduction efficiency was measured using a fluorescence plate reader. CAR expression in the human primary breast carcinomas and cell lines was also evaluated by IHC.ResultsWe observed membranous CAR, p120-catenin and E-cadherin expression in IDC. In ILC, we observed cytoplasmic expression of CAR and p120-catenin, with absent E-cadherin. Adenovirus effectively transduced high-CAR IDC cell lines, at MOIs as low as 12.5. Ad-GFP showed similar transduction as DfAd-GFP in high-CAR IDC cell lines. Conversely, Ad-GFP transduction of ILC cell lines was observed only at MOIs of 50 and 100. Furthermore, Ad-GFP did not transduce CAR-negative IDC cell lines even at MOIs greater than 100. Liposome encapsulation (DfAd-GFP) improved transduction efficiency 4-fold in ILC and 17-fold in CAR-negative IDC cell lines.ConclusionThe present study demonstrates that oncolytic adenoviral therapy is less effective in ILC than IDC due to differences in spatial CAR expression. Liposome-enhanced delivery may be beneficial for patients with ILC and tumors with low or negative CAR expression to improve adenoviral therapeutic effectiveness.

Evaluation of engraftment and growth dynamics of orthotopic and heterotopic in vivo models of human breast cancer

Purpose of the study. This work was to assess the engraftment and growth dynamics of breast cancer xenografts during orthotopic and subcutaneous injection using various types of biological material, as well as to develop an adequate model of breast cancer for further research.Materials and methods. We used a disaggregated fragment of a tumor obtained from the patient, a certified breast cancer cell line VT20 – human breast carcinoma; a primary human breast carcinoma cell line. Female immunodeficient mice of the Balb/c Nude line in the amount of 36 animals were used as recipient animals. The subcutaneous and orthotopic models of breast cancer were developed in this project. Tumor growth was observed for 28 days from the moment of injection and tumor nodes were measured 2 times a week until the end of the experiment. Results were assessed using medians and percentiles. The nonparametric Mann-Whitney test was used to assess the significance of differences.Results. The dynamics of the growth of tumor cells when injected into various sites was determined in the process of this work. The most successful in terms of a subcutaneous injection was the injection of tumor cells of the certified VT20 line. By the end of the experiment, the median tumor node of this group was 100.32 mm³. The analysis revealed tumor dynamics with orthotopic injection of tumor material, and the median volume of the tumor node in the group with the passport culture cell VT20 and the primary culture cell reached the same value – 149.22 and 148.25. mm³. It was found that both the cell line and the cell suspension were injected into tumor nodes that reached a significantly larger volume when injected orthotopically.Conclusion. We have obtained a tumor model of breast cancer using various methods of material implantation and with the possibility of further use in testing new pharmacological substances.

Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

Na+,HCO3--cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3--cotransport with human breast cancer metastasis. We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3--cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3--cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth.

Detection of Cellular Senescence Reveals the Existence of Senescent Tumor Cells within Invasive Breast Carcinomas and Related Metastases

Oncogene-induced senescence is thought to constitute a barrier to carcinogenesis by arresting cells at risk of malignant transformation. However, numerous findings suggest that senescent cells may conversely promote tumor growth and metastatic progression, for example, through the senescence-associated secretory phenotype (SASP) they produce. Here, we investigated the degree to which senescent tumor cells exist within untreated human primary breast carcinomas and whether the presence of senescent cancer cells in primary tumors is recapitulated in their matched lymph node metastases. For the detection of senescence, we used SA-β-galactosidase (SA-β-gal) staining and other senescence markers such as Ki67, p21, p53, and p16. In patients with invasive luminal A and B breast carcinomas, we found broad similarities in the appearance of cancer cells between primary tumors and their corresponding metastases. Analysis of lymph nodes from patients with other breast cancer subtypes also revealed senescent tumor cells within metastatic lesions. Collectively, our findings show that senescent tumor cells exist within primary breast carcinomas and metastatic lesions. These results suggest a potential role for senescent breast tumor cells during metastatic progression and raise the question as to whether the targeting of senescent tumor cells with anti-senescent drugs might represent a novel avenue for improved treatment of breast and other cancers.

Chromatin accessibility landscape and active transcription factors in primary human invasive lobular and ductal breast carcinomas

BackgroundInvasive lobular breast carcinoma (ILC), the second most prevalent histological subtype of breast cancer, exhibits unique molecular features compared with the more common invasive ductal carcinoma (IDC). While genomic and transcriptomic features of ILC and IDC have been characterized, genome-wide chromatin accessibility pattern differences between ILC and IDC remain largely unexplored.MethodsHere, we characterized tumor-intrinsic chromatin accessibility differences between ILC and IDC using primary tumors from The Cancer Genome Atlas (TCGA) breast cancer assay for transposase-accessible chromatin with sequencing (ATAC-seq) dataset.ResultsWe identified distinct patterns of genome-wide chromatin accessibility in ILC and IDC. Inferred patient-specific transcription factor (TF) motif activities revealed regulatory differences between and within ILC and IDC tumors. EGR1, RUNX3, TP63, STAT6, SOX family, and TEAD family TFs were higher in ILC, while ATF4, PBX3, SPDEF, PITX family, and FOX family TFs were higher in IDC.ConclusionsThis study reveals the distinct epigenomic features of ILC and IDC and the active TFs driving cancer progression that may provide valuable information on patient prognosis.

XAF1 drives apoptotic switch of endoplasmic reticulum stress response through destabilization of GRP78 and CHIP

X-linked inhibitor of apoptosis-associated factor-1 (XAF1) is a stress-inducible tumor suppressor that is commonly inactivated in many human cancers. Despite accumulating evidence for the pro-apoptotic role for XAF1 under various stressful conditions, its involvement in endoplasmic reticulum (ER) stress response remains undefined. Here, we report that XAF1 increases cell sensitivity to ER stress and acts as a molecular switch in unfolded protein response (UPR)-mediated cell-fate decisions favoring apoptosis over adaptive autophagy. Mechanistically, XAF1 interacts with and destabilizes ER stress sensor GRP78 through the assembly of zinc finger protein 313 (ZNF313)-mediated destruction complex. Moreover, XAF1 expression is activated through PERK-Nrf2 signaling and destabilizes C-terminus of Hsc70-interacting protein (CHIP) ubiquitin E3 ligase, thereby blocking CHIP-mediated K63-linked ubiquitination and subsequent phosphorylation of inositol-required enzyme-1α (IRE1α) that is involved in in the adaptive ER stress response. In tumor xenograft assays, XAF1−/− tumors display substantially lower regression compared to XAF1+/+ tumors in response to cytotoxic dose of ER stress inducer. XAF1 and GRP78 expression show an inverse correlation in human cancer cell lines and primary breast carcinomas. Collectively this study uncovers an important role for XAF1 as a linchpin to govern the sensitivity to ER stress and the outcomes of UPR signaling, illuminating the mechanistic consequence of XAF1 inactivation in tumorigenesis.

Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival.

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3- cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3- cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3- cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3- cotransport associates with severe breast cancer: enlarged CO2/HCO3--dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3--dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3- cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

Evaluation of breast cancer stem cells in human primary breast carcinoma and their role in aggressive behavior of the disease

To delineate the underlying molecular mechanisms responsible for the intratumoral enrichment of breast cancer stem cells (BCSCs) in aggressive breast tumors, we evaluated the frequency and characteristics of BCSCs within the tumor tissue in primary human breast carcinomas. We assessed the expression profiles of various genes in cancer cells (CC) and stromal cells (SC) from these tumors to delineate the role played by the cellular niche in de novo origin or expansion of intra-tumoral cancer stem cells (CSC). The study included primary tumor and adjacent normal breast tissue specimens from chemotherapy-naïve breast carcinoma patients. The BCSCs, identified as Lin-CD44+CD24- and aldehyde dehydrogenase 1 A1 positive, were enumerated. The flow-cytometrically sorted stromal, and CC were processed for gene expression profiling using a custom-designed polymerase chain reaction array of genes known to facilitate disease progression. The frequency of BCSCs within the tumor mass correlated significantly with histopathological and molecular grades of tumors, indicating a direct relationship of BCSC with the aggressive behavior of breast cancer. Further, a significantly increased expression of the genes associated with growth factors, cytokines and matricellular proteins in tumors were found in high BCSCs compared to Lo-BCSC tumors, suggesting the possible contribution of stromal and CC in an intratumoral expansion of CSCs. Similarly, a significant upregulation of genes associated with hypoxia and angiogenesis in Hi-BCSCs tumors further supported the role of a hypoxic environment. Overall, the findings suggest the molecular crosstalk between SC and CC potentially (directly or indirectly) contributes to the expansion of CSC. The current study highlights the importance of CSC as a potential future predictive/prognostic marker for aggressive breast cancer. The present study predicts the potential risk stratification based on the frequency of BCSCs in primary breast tumors and existing prognostic factors.

Differential expression of Oct3/4 in human breast cancer and normal tissues.

Oct3/4, a transcription factor specifically expressed in mammalian totipotent embryonic stem and germ cells, has a critical role in the regulation and maintenance of pluripotency and self-renewal. However, reactivation of Oct3/4 expression is observed in several human breast cancer cell lines, but not in non‑malignant cells. To examine Oct3/4 expression in human primary breast carcinomas and normal breast tissues, we obtained breast tumor tissues from 28patients and normal breast tissues from 9women. According to quantitative polymerase chain reaction, all of the tumor tissues, irrespective of tumor type or clinicopathological status, expressed Oct3/4 mRNA at 10- to100- fold higher levels than that in the normal breast tissues. Expression of the Oct3/4 protein in tumors was confirmed by western blot analysis and immunofluorescent staining. Additionally, rapid amplification of cDNA ends and DNA sequencing revealed expression of multiple Oct4 gene transcripts from chromosome6 (POU5F1) in normal breast tissues and the non‑malignant breast epithelial cell line MCF‑10A; by contrast, the breast tumors and malignant breast cancer cell line MCF‑7 predominantly expressed transcripts of an Oct4-like gene (POU5F1B) from chromosome8, which was termed Oct3 in the current study. The deduced amino acid sequences of full-length Oct3 and Oct4 are 96% identical. The findings of the current study indicated that Oct3, rather than Oct4, may serve as a novel clinical marker and a potential target for gene-specific therapy of breast cancer.

Insertional mutagenesis in a HER2-positive breast cancer model reveals ERAS as a driver of cancer and therapy resistance

Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS. The CIS most strongly associated with the ErbB2-transgenic genotype was the locus containing Eras (ES cell-expressed Ras), a constitutively active RAS-family GTPase. We show that upon expression, Eras acts as a potent oncogenic driver through hyperactivation of the PI3K/AKT pathway, in contrast to other RAS proteins that signal primarily via the MAPK/ERK pathway and require upstream activation or activating mutations to induce signaling. We additionally show that ERAS synergistically enhances HER2-induced tumorigenesis and, in this role, can functionally replace ERBB3/HER3 by acting as a more powerful activator of PI3K/AKT signaling. Although previously reported as pseudogene in humans, we observed ERAS RNA and protein expression in a substantial subset of human primary breast carcinomas. Importantly, we show that ERAS induces primary resistance to the widely used HER2-targeting drugs Trastuzumab (Herceptin) and Lapatinib (Tykerb/Tyverb) in vivo, and is involved in acquired resistance via selective upregulation during treatment in vitro, indicating that ERAS may serve as a novel clinical biomarker for PI3K/AKT pathway hyperactivation and HER2-targeted therapy resistance.

Abstract P5-05-03: Upregulated purinergic signaling enhances cell proliferation in human and murine breast carcinomas

Abstract The composition of the extracellular tumor microenvironment differs from that of most other tissues and is thought to provide cancer cells with a growth and survival advantage compared to normal cells. In solid tumors, the extracellular concentration of ATP can be elevated to ~100 µm and extracellular pH can be as low as 6.5. In the current project, we investigate the consequences of purinergic signaling in human and murine breast carcinomas: we study intracellular Ca2+ signals and associated changes in cell proliferation during stimulation with extracellular nucleotides. We employ biopsies of human and murine primary breast carcinomas and compare them with matched normal breast tissue. Human biopsies are obtained with written informed consent from patients undergoing breast conserving surgery at Aarhus University Hospital or Regional Hospital Randers in Denmark. Murine biopsies are from mice overexpressing unactivated ErbB2 specifically in the breast tissue. We isolate epithelial organoids (~150 µm diameter) from tissue biopsies by partial digestion with collagenase III. Organoids loaded with the Ca2+-sensitive fluorophore Fura-2 are studied by fluorescence microscopy. In separate experiments, cell proliferation is quantified by detecting newly synthesized DNA using immunofluorescence imaging of organoids incubated with the thymidine analogue bromodeoxyuridine (BrdU). We find that intracellular Ca2+ responses during stimulation with extracellular ATP are elevated 2- to 10-fold in breast carcinomas from mice and humans, respectively, compared to matched normal breast tissue. We observe similar differences between breast cancer tissue and normal breast tissue in response to stimulation with the P2Y2/P2Y4-agonist UTP, whereas virtually no rise in the intracellular concentration of Ca2+ is observed in response to the P2X7-agonist 3'-O-(4-benzoyl)benzoyl-ATP. Application of cyclopiazonic acid – an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase – also cause exaggerated intracellular Ca2+ responses in breast cancer compared to normal breast tissue. Consistent with the elevated Ca2+ responses, stimulation with 100 µm ATP or 100 µm UTP increases the rate of cell proliferation (i.e., fraction of BrdU-positive cells) by ~2-fold in the breast cancer tissue. In conclusion, we find that purinergic signaling is upregulated in human and murine breast carcinomas compared to normal breast tissue. Activation of purinergic receptors – most likely P2Y2 and/or P2Y4 – enhances cell proliferation in breast cancer tissue. We propose that the high ATP levels in the tumor microenvironment promote breast cancer development or progression and that the associated signaling pathways represent promising targets for therapy. Citation Format: Mele M, McWhan K, Henningsen M, Vahl P, Jensen V, Johansen T, Pedersen H, Christiansen P, Bødtkjer E. Upregulated purinergic signaling enhances cell proliferation in human and murine breast carcinomas [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-03.

1541P - Presence of tumor-specific cytolytic T cells in human primary breast carcinoma: consequences for immunotherapy

Immunotherapy through stimulatory antibodies targeting the CTLA-4 or PD-1 pathways has a demonstrated clinical efficacy in a fraction of patients with various cancers. It is likely that the main immune effectors of these therapies are CD8+ cytolytic T lymphocytes (CTL) recognizing tumor-specific antigens. Highly antigenic tumors such as metastatic melanomas are often immunogenic, i.e. they stimulate spontaneous anti-tumor CTL responses. This immunogenicity, of which the presence of tumor-infiltrating T cells (TILs) is probably a surrogate marker, might be a predictive marker for clinical benefit to immunostimulatory antibodies. The immunogenicity of primary breast carcinomas for CD8+ T cells has not been studied, but the amounts of TILs have been positively correlated with patients' survival. Here we wished to obtain evidence for the presence of tumor-specific CD8+ T cells in TILs from primary breast carcinomas. From 5 tumors (2 ER+/HER2-, 2 ER+/HER2+, 1 ER-/HER2-) we isolated TILs and derived a random set of ±100 CD8+ T cell clones that we screened for recognition of candidate tumor-specific antigenic peptides selected through tumor exome sequencing and gene expression profiling. These peptides were encoded either by mutated genes or by cancer-germline genes. For 4 tumors, we screened 61-142 T cell clonotypes for recognition of 23-61 candidate mutated peptides, without any positive result. For the last tumor (ER+/HER2-), 6 out of 57 T cell clonotypes recognized 4 out of 109 candidate mutated peptides but not the corresponding wild-type peptides. This tumor contained more mutations than the other four, and displayed microsatellite instability. Some human primary breast carcinomas are immunogenic, as one tumor contained at least 10% of tumor-specific cells among the CD8+ TILs. Our observation corroborates the association between high mutation burden and CTL response to mutated tumor antigens. The presence of tumor-specific CD8+ suggests that the corresponding patient could benefit from the currently used immunostimulatory antibodies.

Abstract P4-04-08: Presence of tumor-specific cytolytic T cells in human primary breast carcinoma: Consequences for immunotherapy

Abstract Background: Immunotherapy through stimulatory antibodies targeting the CTLA-4 or PD-1 pathways has a clear clinical efficacy in a fraction of patients with various cancers. It is likely that the main immune effectors of these therapies are CD8+ cytolytic T lymphocytes (CTL) recognizing tumor-specific antigens. The antigenicity of human tumors has been demonstrated with studies conducted mostly on melanomas. However the genetic mechanisms leading to antigenicity, notably point mutations in the tumor cells, apply to all cancer types. Thus primary breast carcinoma cells do certainly bear tumor-specific antigens, even though the extent of this antigenicity is unknown. Most melanomas, which are highly antigenic tumors, are also immunogenic, i.e. they stimulate spontaneous anti-tumor CTL responses. This immunogenicity, of which the presence of tumor-infiltrating T cells (TILs) is probably a surrogate marker, might be a predictive marker for clinical benefit to immunostimulatory antibodies. Whether primary breast carcinomas are immunogenic is not known, mainly due the absence of autologous tumor cell lines to analyze patients' T cells. However even in the absence of T-cell aimed immunotherapy the amounts of TILs have been positively correlated with patients' survival. Here we wished to obtain evidence for the presence of tumor-specific CD8+ T cells in TILs from primary breast carcinomas. Methods: From each tumor we isolated TILs and derived a random set of ±100 CD8+ clones maintained in culture by stimulation with anti-CD3 antibodies, thus irrespective of their antigenic specificity. We screened these clones for recognition of tumor-specific antigens present on the autologous tumor. In the absence of autologous tumor lines we restricted our analysis to mutated antigens selected on the basis of tumor exome sequencing and gene expression profiling. Indels and non-synonymous base substitutions were selected to synthesize candidate mutated peptides. Results: Thus far we have analyzed two hormone receptor-positive HER2-negative primary carcinomas. For one patient we screened 144 T cell clones for recognition of 40 candidate mutated peptides, without any positive result. For the other patient, 6 out of 98 T cell clones recognized 4 out of 119 candidate mutated peptides. Two peptides were recognized by two different T cell clones, i.e. with different T cell receptor sequences. These 4 'antigenic' mutations appear to be passenger, i.e. the four genes have a low published mutation frequency. Conclusions: We conclude that some human primary breast carcinomas are immunogenic, as one tumor contained at least 6% of tumor-specific T cells among the CD8+ TILs. It suggests that the corresponding patient could benefit from the currently used immunostimulatory antibodies. More work is required to understand the reasons for the negative results in the first patient. We are pursuing the work on 2 HER2-positive and 2 triple-negative tumors, in which TILs are better correlated with prognosis. Our results warrant more investigations on the activation or inhibition of tumor-specific T cells at early stages of human breast cancer development. Citation Format: Schröder DJ, Bricard O, Hames G, Remy N, Carrasco J, Canon J-L, Berlière M, Galant C, Coulie PG. Presence of tumor-specific cytolytic T cells in human primary breast carcinoma: Consequences for immunotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-08.

Abstract 4161: Differential expression of VEGFR2 Protein in HER2 positive primary human breast cancer: Potential relevance to newer anti-angiogenic therapies

Abstract Previously, we demonstrated immunohistochemical expression of VEGFR2 protein and vascular phenotypes in human breast carcinomas (Holzer, Nasir et al AACR 2014). Here, we carried out a large-scale immunopathologic analysis of tumor vascular density and differential expression of VEGFR2 protein in various subsets of primary human breast carcinomas from 186 females (Mean age: 59 years; range 33-88 years). Sections from a primary breast cancer tissue microarray (Yale University), were stained for a sensitive tumor vascular marker (CD34) and for VEGFR2 protein using a novel VEGFR2 IHC assay developed and standardized in our laboratory (Holzer, Nasir et al PLOS One 2013). Discrete VEGFR2+ and CD34+ tumor vessels were counted in each analyzable TMA core by an experienced breast pathologist, excluding any suboptimal/inadequate cores or any areas of tumor necrosis. Histologic subtypes of primary breast carcinomas included invasive ductal, lobular, mixed ductal-lobular and colloid (N = 139, 22, 18, 7) respectively. Based on hormone receptor (HR) and Her2 status, cases were grouped into HR+, Her2+ and triple-negative subsets. Pathologic assessments of vascular density (CD34 counts) and imunohistochemical expression of VEGFR2 protein were made only in the viable invasive carcinoma tissue. The observed CD34+ and VEGFR2+ tumor vascular counts in individual cases were heterogeneous, ranging from high vascular density and high VEGFR2 expression to minimal vascular density and/or minimal or no VEGFR2 expression. Among all histologies, mean CD34+ and VEGFR2+ tumor vascular counts were 11 and 3.4 per tumor TMA core respectively. Eighty-nine of 186 (48%) cases had >10 CD34+ tumor vessels, while 97/186 (52%) had fewer CD34+ vessels in each TMA core. Of 169 analyzable cores in the VEGFR2 stained TMA, 90 (53%) showed 1 to 5 VEGFR2+ tumor vessels/TMA core, while 42/169 (25%) cores had no VEGFR2+ tumor vessels. Thirteen of 169 (8%) cases also showed tumor cell (cytoplasmic and/or membrane) expression of VEGFR2 protein. Triple-negative breast cancers (TNBCs) appeared to be less vascular (Mean VD = 9.8, range 0-34) than other breast cancer subtypes. Overall, VEGFR2+ tumor vessel counts were significantly higher in Her2+ as compared to HR+ (p = 0.03) and TNBC (p = 0.01) tissues. Compared to Her2- cases, Her2+ breast cancers had higher VEGFR2+ tumor vessel counts (p = 0.006). These data provide insights about tumor vascular density and vascular VEGFR2 expression in primary human breast cancer subtypes, especially Her2 positive breast cancer, and offer a pathobiologic hypothesis for the patterns of clinical response of breast cancer patients to newer anti-angiogenic therapies. Citation Format: Aejaz Nasir, Timothy R. Holzer, Michael Man, Laura E. Benjamin, Allen S. Melemed, Andrerw E. Schade. Differential expression of VEGFR2 Protein in HER2 positive primary human breast cancer: Potential relevance to newer anti-angiogenic therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4161. doi:10.1158/1538-7445.AM2015-4161

Abstract 154: Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis

Abstract Background: Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. TGFβ signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Methods: Genome-wide lncRNA profile was performed using NCode™ Mouse Noncoding Microarray chip from Life Technologies, which contains 10,802 lncRNAs and 25,178 protein-coding genes. Total RNA was isolated from mouse mammary epithelial NMuMG cells, which had been treated with TGFβ for 0, 12 and 24 hours, and was used to hybridize the microarray. The role of a top TGFβ-upregulated lncRNA, lncRNA-HIT, in EMT and metastasis was further characterized in cell culture and orthotopic breast cancer models Results: Among 10,802 lnRNAs profiled, 680 were up-regulated and 633 were down-regulated during TGFβ-induced EMT, respectively. Further, we identify that lncRNA-HIT (HOXA antisense transcript induced by TGFβ) mediates TGFβ function, i.e., depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth and metastasis. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Conclusions: These data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers. Citation Format: Edward Richards, Gu Zhang, Jennifer Permuth-Wey, Zhu-Peng Li, Sridevi Challa, William Kong, Dan Su, Domenico Coppola, Wei-min Mao, Thomas Sellers, Jin Q. Cheng. Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2015-154

Long Non-coding RNAs (LncRNA) Regulated by Transforming Growth Factor (TGF) β: LncRNA-HIT-MEDIATED TGFβ-INDUCED EPITHELIAL TO MESENCHYMAL TRANSITION IN MAMMARY EPITHELIA

Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Highly magnetic Fe2O3 nanoparticles synthesized by laser pyrolysis used for biological and heat transfer applications

γFe2O3-based nanoparticles were synthesized by laser pyrolysis using various optimized Fe(CO)5, O2 and C2H4 flow ratios in the reactive mixture, and different laser power values. Depending on particular conditions, two different iron oxide-based nanoparticles (MNPs) were synthesized, with a hydrophilic or hydrophobic behavior, both presenting a high magnetization saturation (around 70emu/g). TEM, EDX, XRD and magnetic analyses were performed for a comprehensive characterization. The raw powders were successfully dispersed in aqueous media using l-DOPA as stabilizing agent. Dispersed samples, with or without stabilization agents, have been tested and DLS measurements proved their good stability, with the hydrodynamic diameter varying between 70 and 150nm when the stabilizing agent was used. Thermal conductivity and viscosity tests on l-DOPA-functionalized MNPs suspensions reveal the increasing (up to 40%) of their thermal conductivity, accompanied by a viscosity increase of only 5%, validating them as thermal transfer fluids. Water-based nanoparticle dispersions and also those stabilized with l-DOPA proved a good biocompatibility, as demonstrated by a preliminary in vitro study on mouse primary leukocytes and human breast carcinoma cell line MCF-7; although ingested by the investigated cells, MNPs do not decrease cellular viability and proliferation.

The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype.

BackgroundCancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia.MethodsImmunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting.ResultsWe demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population.ConclusionsOur results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-734) contains supplementary material, which is available to authorized users.

Na+,HCO3- -cotransport is functionally upregulated during human breast carcinogenesis and required for the inverted pH gradient across the plasma membrane.

Metabolic and biochemical changes during breast carcinogenesis enhance cellular acid production. Extrusion of the acid load from the cancer cells raises intracellular pH, while it decreases extracellular pH creating an inverted pH gradient across the plasma membrane compared to normal cells and promoting cancer cell metabolism, proliferation, migration, and invasion. We investigated the effects of breast carcinogenesis on the mechanisms of cellular pH control using multicellular epithelial organoids freshly isolated from human primary breast carcinomas and matched normal breast tissue. Intracellular pH was measured by fluorescence microscopy, while protein expression was investigated by immunofluorescence imaging and immunoblotting. We found that cellular net acid extrusion increased during human breast carcinogenesis due to enhanced Na(+),HCO3 (-)-cotransport, which created an alkaline shift (~0.3 units of magnitude) in steady-state intracellular pH of human primary breast carcinomas compared to normal breast tissue. Na(+)/H(+)-exchange activity and steady-state intracellular pH in the absence of CO2/HCO3 (-) were practically unaffected by breast carcinogenesis. These effects were evident under both acidic (pH 6.8, representative of the tumor microenvironment) and physiological (pH 7.4) extracellular conditions. Protein expression of the Na(+),HCO3 (-)-cotransporter NBCn1 (SLC4A7), which has been linked to breast cancer susceptibility in multiple genome-wide association studies, was twofold higher in human breast carcinomas compared to matched normal breast tissue. Protein expression of the Na(+)/H(+)-exchanger NHE1 (SLC9A1) was markedly less affected. We propose that upregulated NBCn1 during human breast carcinogenesis contributes to the characteristic acid distribution within human breast carcinomas and thereby plays a pathophysiological role for breast cancer development and progression.

Microenvironmental stimuli affect Endothelin-1 signaling responsible for invasiveness and osteomimicry of bone metastasis from breast cancer

The present study was undertaken to clarify the function(s) of Endothelin-1 and its receptors ETAR and ETBR in osteolytic-bone metastasis from breast cancer, and their regulation by hepatocyte and transforming growth factors (HGF, TGF-β) and hypoxia. The aim was to evaluate the adaptability of bone metastasis to microenvironmental stimuli through Endothelin-1-mediated epithelial-mesenchymal transition (EMT), or the reverse process MET, and through osteomimicry possible key features for bone colonization. We compared low (MCF-7) and high (MDA-MB231) invasive-breast carcinoma cells, and 1833-bone metastatic clone, with human pair-matched primary breast-carcinomas and bone metastases. Parental MDA-MB231 and the derived 1833-clone responded oppositely to the stimuli. In 1833 cells, TGF-β and hypoxia increased Endothelin-1 release, altogether reducing invasiveness important for engraftment, while Endothelin-1 enhanced MDA-MB231 cell invasiveness. The Endothelin-1-autocrine loop contributed to the cooperation of intracellular-signaling pathways and extracellular stimuli triggering MET in 1833 cells, and EMT in MDA-MB231 cells. Only in 1833 cells, HGF negatively influenced transactivation and release of Endothelin-1, suggesting a temporal sequence of these stimuli with an initial role of HGF-triggered Wnt/β-catenin pathway in metastatization. Then, Endothelin-1/ETAR conferred MET and osteomimetic phenotypes, with Runt-related transcription factor 2 activation and metalloproteinase 9 expression, contributing to colonization and osteolysis. Findings with human pair-matched primary ductal carcinomas and bone metastases gave a translational significance to the molecular study. Endothelin-1, ETAR and ETBR correlated with the acquisition of malignant potential, because of high expression already in the in situ carcinoma. These molecular markers might be used as predictive index of aggressive behavior and invasive/metastatic phenotype.