Human Breast Tumor Specimens Research Articles

Elucidating the Role of MicroRNA-18a in Propelling a Hybrid Epithelial-Mesenchymal Phenotype and Driving Malignant Progression in ER-Negative Breast Cancer.

Epigenetic alterations that lead to differential expression of microRNAs (miRNAs/miR) are known to regulate tumour cell states, epithelial-mesenchymal transition (EMT) and the progression to metastasis in breast cancer. This study explores the key contribution of miRNA-18a in mediating a hybrid E/M cell state that is pivotal to the malignant transformation and tumour progression in the aggressive ER-negative subtype of breast cancer. The expression status and associated effects of miR-18a were evaluated in patient-derived breast tumour samples in combination with gene expression data from public datasets, and further validated in in vitro and in vivo breast cancer model systems. The clinical relevance of the study findings was corroborated against human breast tumour specimens (n = 446 patients). The down-regulated expression of miR-18a observed in ER-negative tumours was found to drive the enrichment of hybrid epithelial/mesenchymal (E/M) cells with luminal attributes, enhanced traits of migration, stemness, drug-resistance and immunosuppression. Further analysis of the miR-18a targets highlighted possible hypoxia-inducible factor 1-alpha (HIF-1α)-mediated signalling in these tumours. This is a foremost report that validates the dual role of miR-18a in breast cancer that is subtype-specific based on hormone receptor expression. The study also features a novel association of low miR-18a levels and subsequent enrichment of hybrid E/M cells, increased migration and stemness in a subgroup of ER-negative tumours that may be attributed to HIF-1α mediated signalling. The results highlight the possibility of stratifying the ER-negative disease into clinically relevant groups by analysing miRNA signatures.

Optimal Phased-Array Signal Combination For Polyunsaturated Fatty Acids Measurement In Breast Cancer Using Multiple Quantum Coherence MR Spectroscopy At 3T

Polyunsaturated fatty acid (PUFA), a key marker in breast cancer, is non-invasively quantifiable using multiple quantum coherence (MQC) magnetic resonance spectroscopy (MRS) at the expense of losing half of the signal. Signal combination for phased array coils provides potential pathways to enhance the signal to noise ratio (SNR), with current algorithms developed for conventional brain MRS. Since PUFA spectra and the biochemical environment in the breast deviate significantly from those in the brain, we set out to identify the optimal algorithm for PUFA in breast cancer. Combination algorithms were compared using PUFA spectra from 17 human breast tumour specimens, 15 healthy female volunteers, and 5 patients with breast cancer on a clinical 3 T MRI scanner. Adaptively Optimised Combination (AOC) yielded the maximum SNR improvement in specimens (median, 39.5%; interquartile range: 35.5–53.2%, p < 0.05), volunteers (82.4 ± 37.4%, p < 0.001), and patients (median, 61%; range: 34–105%, p < 0.05), while independent from voxel volume (rho = 0.125, p = 0.632), PUFA content (rho = 0.256, p = 0.320) or water/fat ratio (rho = 0.353, p = 0.165). Using AOC, acquisition in patients is 1.5 times faster compared to non-noise decorrelated algorithms. Therefore, AOC is the most suitable current algorithm to improve SNR or accelerate the acquisition of PUFA MRS from breast in a clinical setting.

The Protease Activated Receptor2 Promotes Rab5a Mediated Generation of Pro-metastatic Microvesicles

Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/functional behavior of cells to break attachment and migrate to distant body parts. Cancer cell-secreted microvesicles (MVs) contribute immensely in disease propagation. These nano-vesicles, generated from plasma membrane outward budding are taken up by nearby healthy cells thereby inducing phenotypic alterations in those recipient cells. Protease activated receptor 2 (PAR2), activated by trypsin, also contributes to cancer progression by increasing metastasis, angiogenesis etc. Here, we report that PAR2 activation promotes pro-metastatic MVs generation from human breast cancer cell line, MDA-MB-231. Rab5a, located at the plasma membrane plays vital roles in MVs biogenesis. We show that PAR2 stimulation promotes AKT phosphorylation which activates Rab5a by converting inactive Rab5a-GDP to active Rab5a-GTP. Active Rab5a polymerizes actin which critically regulates MVs shedding. Not only MVs generation, has this Rab5a activation also promoted cell migration and invasion. We reveal that Rab5a is over-expressed in human breast tumor specimen and contributes MVs generation in those patients. The involvement of p38 MAPK in MVs-induced cell metastasis has also been highlighted in the present study. Blockade of Rab5a activation can be a potential therapeutic approach to restrict MVs shedding and associated breast cancer metastasis.

Alternative Polyadenylation of PRELID1 Regulates Mitochondrial ROS Signaling and Cancer Outcomes.

Disruption of posttranscriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the PRELID1 mRNA that enhances its steady-state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it has been demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell type-specific manner. Modulation of PRELID1 expression, including its posttranscriptional control, appears to be a common stress response across different cancer types. These data reveal that PRELID1 mRNA processing is an important regulator of cell type-specific responses to stress used by multiple cancers and is associated with patient outcomes.Implications: This study suggests that the regulation of PRELID1 expression, by APA and other mechanisms, plays a role in mitochondrial ROS signaling and represents a novel prognostic factor and therapeutic target in cancer. Mol Cancer Res; 15(12); 1741-51. ©2017 AACR.

Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy

ABSTRACTFormation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis.

Abstract 4921: The adhesion protein JAM-A is important for maintenance of tumor dormancy in breast cancer cells, and JAM-A high-dormant cells have stem cell characteristics

Abstract Breast cancer cells which metastasize to bone marrow can remain dormant and survive multiple rounds of adjuvant chemotherapy. The re-awakening of dormant populations, particularly in estrogen receptor (ER)-positive patients, has been associated with tumor cell proliferation and poor patient outcomes. Tumor dormancy can be modelled in vitro by exposing cancer cells on fibronectin-coated plates to bFGF-2, a mammary differentiation factor abundant in the bone marrow stroma, which causes partial re-differentiation, cell spreading and re-expression of integrin-α5β1. Ligation of integrin-α5β1 by fibronectin and activation of the PI3K pathway both contribute to dormant cell survival. Our study has uncovered a novel potential role for the adhesion protein Junctional Adhesion Molecule-A (JAM-A) in tumor dormancy. JAM-A overexpression has previously been linked to increased risk of metastasis in breast cancer patients, and its expression regulates the angiogenic functions of bFGF-2. Furthermore, since loss of JAM-A causes down-regulation of the α5β1 downstream effector FAK, we hypothesized that JAM-A is required for maintenance of breast cancer cell dormancy. To investigate this, JAM-A was transiently silenced using siRNA in MCF-7 breast cancer cells grown on fibronectin-coated plates and exposed to FGF-2. JAM-silenced cells exhibited downregulations in the protein expression of integrin-α5β1 and FAK in this model, and failed to exhibit the cortical actin redistribution and morphological spreading which characterize dormant cells. We next investigated a potential relationship between JAM-A expression and pro-inflammatory cytokines which have been linked with the re-awakening of dormant cells in post-menopausal women. Exposure of MCF-7 cells to the pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-8 decreased JAM-A expression in in a concentration-dependent fashion, suggesting that the loss of JAM-A could be associated with re-awakening from tumor dormancy. We are currently probing alterations in JAM-A expression and that of integrin-α5β1 in matched human primary breast tumor specimens and bone marrow metastases. Since JAM-high dormant cells express high mRNA levels of the stemness factors (Sox2, Oct3/4, Nanog) and have high Aldefluor activity, our data also suggest that JAM-A may be required for a stem-like phenotype. To conclude, our data support a model whereby JAM-A is important for the maintenance of dormancy in breast cancer cells in a simulated bone marrow microenvironment, and represents a novel therapeutic target worthy of investigation in breast cancer. Citation Format: Sri Harikrishna Vellanki, Rodrigo G. Cruz, Ann M. Hopkins. The adhesion protein JAM-A is important for maintenance of tumor dormancy in breast cancer cells, and JAM-A high-dormant cells have stem cell characteristics [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 4921. doi:10.1158/1538-7445.AM2017-4921

Heterogeneous perivascular cell coverage affects breast cancer metastasis and response to chemotherapy.

Angiogenesis and co-optive vascular remodeling are prerequisites of solid tumor growth. Vascular heterogeneity, notably perivascular composition, may play a critical role in determining the rate of cancer progression. The contribution of vascular pericyte heterogeneity to cancer progression and therapy response is unknown. Here, we show that angiopoietin-2 (Ang2) orchestrates pericyte heterogeneity in breast cancer with an effect on metastatic disease and response to chemotherapy. Using multispectral imaging of human breast tumor specimens, we report that perivascular composition, as defined by the ratio of PDGFRβ- and desmin+ pericytes, provides information about the response to epirubicin but not paclitaxel. Using 17 distinct patient-derived breast cancer xenografts, we demonstrate a cancer cell-derived influence on stromal Ang2 production and a cancer cell-defined control over tumor vasculature and perivascular heterogeneity. The aggressive features of tumors and their distinct response to therapies may thus emerge by the cancer cell-defined engagement of distinct and heterogeneous angiogenic programs.

Multiplexed Molecular Imaging of Biomarker-Targeted SERS Nanoparticles on Fresh Tissue Specimens with Channel-Compressed Spectrometry.

Biomarker-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have been explored as a viable option for targeting and imaging multiple cell-surface protein biomarkers of cancer. While it has been demonstrated that this Raman-encoded molecular imaging (REMI) technology may potentially be used to guide tumor-resection procedures, the REMI strategy would benefit from further improvements in imaging speed. Previous implementations of REMI have utilized 1024 spectral channels (camera pixels) in a commercial spectroscopic CCD to detect the spectral signals from multiplexed SERS NPs, a strategy that enables accurate demultiplexing of the relative concentration of each NP “flavor” within a mixture. Here, we investigate the ability to significantly reduce the number of spectral-collection channels while maintaining accurate imaging and demultiplexing of up to five SERS NP flavors, a strategy that offers the potential for improved imaging speed and/or detection sensitivity in future systems. This strategy was optimized by analyzing the linearity of five multiplexed flavors of SERS NPs topically applied on tissues. The accuracy of this binning approach was then validated by staining tumor xenografts and human breast tumor specimens with a mixture of five NP flavors (four targeted NPs and one untargeted NP) and performing ratiometric imaging of specific vs. nonspecific NP accumulation. We demonstrate that with channel-compressed spectrometry using as few as 16 channels, it is possible to perform REMI with five NP flavors, with < 20% error, at low concentrations (< 10 pM) that are relevant for clinical applications.

Abstract 2865: Dual action glycotherapy for triple negative breast cancer

Abstract Triple negative breast cancer (TNBC; ER-/PR-/HER2-) accounts for 15% of all breast cancer with a disproportionate share of mortality. The patients are younger and pre-menopausal. The cancers are poorly differentiated, and most fall into the basal subgroup of breast cancers. The long-term survival is extremely low because of lack of specific treatment guidelines. Thus, the patients are managed with standard treatment. This causes a high rate of local and systemic relapse, and make patients resistant to existing targeted therapies (endocrine/biologics/adjuvant/neo-adjuvant). Anthracycline/taxane combination therapy and PARP inhibitor are currently being explored but not known what would be the outcome or if the patients would achieve a pathological complete response (pCR). Protein glycosylation has been claimed as an important feature helping cancer cells escaping immune surveillance, facilitate tumor invasion, and increased malignancy with increased tumor burden and poor prognosis as well as enhanced angiogenesis. Based on the glycomics profile of human breast cancer cells and tumor specimen, we have hypothesized that targeting asparagine-linked (N-linked) protein glycosylation would evolve a new generation therapeutic preventing breast tumor progression and eliminating the disease. Consequently, when treated with a homolog of protein N-glycosylation inhibitor tunicamycin the progression of a double negative breast cancer was inhibited significantly (J. Biol. Chem. 286, 29127-29138, 2011). We have now tested a triple negative breast tumor xenograft in athymic nude mice Balb/c (nu/nu) and the result is reduction of tumor progression (∼65% in one week) at tunicamycin concentration of 0.25 mg/Kg given orally twice a week. Human triple negative breast cancer cells (MDA-MB-231/MDA-MB-468) are equally susceptible to tunicamycin action. The cells are arrested in G1 and exhibit ER stress followed by induction of apoptosis mediated by unfolded protein response (upr) signaling. Western blotting and qRT-PCR support increased expression of the ER stress master regulator GRP78. But, the immunofluorescence microscopy could not detect GRP78 on the surface of control or tunicamycin treated cells. The result is identical in cells cultured in the absence of serum. GRP78 fluorescence however, is detectable in cells either fixed with ice-cold methanol or permeabilized with digitonin. We, therefore, conclude that GRP78 is not expressed on the outer-leaflet of the cell surface of the triple negative human breast cancer cells MDA-MB-231. But, its intracellular expression is anti-tumorigenic. Supported in part by grants from NSF-EPSCoR RII Track 1 Grant # EPS-1002410 (DKB) and NIH/NIMHD 2G12MD007583 (KB) Citation Format: Dipak K. Banerjee, Aditi Banerjee, Zhenbo Zhang, Jesus E. Serrano, Eva C. Romero, Neysharie Sanchez, Krishna Baksi. Dual action glycotherapy for triple negative breast cancer. [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 2865.

Endophilin A2 Promotes TNBC Cell Invasion and Tumor Metastasis.

Triple-negative breast cancers (TNBCs) are highly aggressive cancers that lack targeted therapies. However, EGFR is frequently activated in a subset of TNBCs and represents a viable clinical target. Because the endocytic adaptor protein Endophilin A2 (SH3GL1/Endo II) has been implicated in EGFR internalization, we investigated Endo II expression and function in human TNBCs. Endo II expression was high in several TNBC cells compared with normal breast epithelial cells. Stable knockdown (KD) of Endo II was achieved in two TNBC cell lines, and although cell viability was unaffected, defects in receptor-mediated endocytosis were observed. EGFR signaling to Erk and Akt kinases was impaired in Endo II KD cells, and this correlated with reduced rates of EGFR internalization and cell motility. Endo II KD cells also displayed defects in three dimensional (3D) cell invasion, and this correlated with impaired extracellular matrix degradation and internalization of MT1-MMP. Endo II silencing also caused a significant reduction in TNBC tumor growth and lung metastasis in mammary orthotopic tumor xenograft assays. In human breast tumor specimens, Endo II expression was highest in TNBC tumors compared with other subtypes, and at the level of gene expression, high Endo II was associated with reduced relapse-free survival in patients with basal-like breast cancers. Together, these results identify a positive role for Endo II in TNBC tumor metastasis and a potential link with poor prognosis. Endophilin A2 and related adaptor proteins represent important signaling hubs to target in metastatic cancers.

Signs analysis and clinical assessment: phase-contrast computed tomography of human breast tumours.

PurposeTo analyse the diagnostic signs present in slices of human breast tumour specimens using synchrotron radiation phase-contrast imaging computed tomography (PCI-CT) for the first time and assess the feasibility of this technique for clinical applications.Materials and MethodsThe ethics committee of our university and relevant clinical hospital approved this prospective study, and written informed consent was obtained from all patients. PCI-CT of human breast tumour specimens with synchrotron radiation was performed at the Shanghai Synchrotron Radiation Facility (SSRF). A total of 14 specimens of early-stage carcinomas and 8 specimens of adenomas were enrolled. Based on raw data reconstruction, the diagnostic signs present in the slices were analysed and correlated with histopathology. We proposed a criterion for clinical diagnosis according to the evaluated signs and the Breast Imaging Reporting and Data System (BI-RADS) for reference. The criterion was then assessed by clinicians in a double-blind method. Finally, descriptive statistics were evaluated, depending on the assessment results.ResultsThe 14 carcinoma specimens and 8 adenoma specimens were diagnosed as malignant and benign tumours, respectively. The total coincidence rate was 100%.ConclusionOur study results demonstrate that the X-ray diagnostic signs observed in the specimen slices and the criterion used for clinical diagnosis were accurate and reliable. The criterion based on signs analysis can be used to differentiate early-stage benign or malignant tumours. As a promising imaging method, PCI-CT can serve as a possible and feasible supplement to BI-RADS in the future.

G protein-coupled estrogen receptor regulates mammary tumorigenesis and metastasis.

The role of 17β-estradiol (E2) in breast cancer development and tumor growth has traditionally been attributed exclusively to the activation of estrogen receptor-α (ERα). Although targeted inhibition of ERα is a successful approach for patients with ERα(+) breast cancer, many patients fail to respond or become resistant to anti-estrogen therapy. The discovery of the G protein-coupled estrogen receptor (GPER) suggested an additional mechanism through which E2 could exert its effects in breast cancer. Studies have demonstrated clinical correlations between GPER expression in human breast tumor specimens and increased tumor size, distant metastasis, and recurrence, as well as established a proliferative role for GPER in vitro; however, direct in vivo evidence has been lacking. To this end, a GPER-null mutation [GPER knockout (KO)] was introduced, through interbreeding, into a widely used transgenic mouse model of mammary tumorigenesis [MMTV-PyMT (PyMT)]. Early tumor development, assessed by the extent of hyperplasia and proliferation, was not different between GPER wild-type/PyMT (WT/PyMT) and those mice harboring the GPER-null mutation (KO/PyMT). However, by 12 to 13 weeks of age, tumors from KO/PyMT mice were smaller with decreased proliferation compared with those from WT/PyMT mice. Furthermore, tumors from the KO/PyMT mice were of histologically lower grade compared with tumors from their WT counterparts, suggesting less aggressive tumors in the KO/PyMT mice. Finally, KO/PyMT mice displayed dramatically fewer lung metastases compared with WT/PyMT mice. Combined, these data provide the first in vivo evidence that GPER plays a critical role in breast tumor growth and distant metastasis. This is the first description of a role for the novel estrogen receptor GPER in breast tumorigenesis and metastasis, demonstrating that it represents a new target in breast cancer diagnosis, prognosis, and therapy.

The serine protease inhibitor elafin maintains normal growth control by opposing the mitogenic effects of neutrophil elastase.

The serine protease inhibitor, elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared to normal human mammary epithelial cells (HMECs). Here, we evaluated the role of elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor deprived HMECs as they enter a quiescent (G0) state, suggesting that elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of elafin compromises the ability of HMECs to maintain G0-arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type elafin, but not elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of elafin to arrest cells in G0. In fact when purified NE was added to elafin knockdown HMECs, these cells demonstrated greater sensitivity to the growth promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples, and immunohistochemical analysis of normal breast tissue revealed robust elafin expression in the mammary epithelium; however, elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE-activity. To address the role of NE in mammary tumorigenesis, we next examined if deregulated NE-activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as elafin, presents an important therapeutic target in breast cancer.

Magnetobiosensors based on viral protein p19 for microRNA determination in cancer cells and tissues.

MicroRNAs (miRs) have emerged as important clinical biomarkers with both diagnostic and prognostic value for relevant diseases, such as cancer. MiRs pose unique challenges for detection and are currently detected by northern blotting, real-time PCR, and microarray techniques. These expensive, complicated, and time-consuming techniques are not feasible for on-site miR determination. In this study, amperometric magnetobiosensors involving RNA-binding viral protein p19 as a selective biorecognition element were developed for miR quantification. The p19-based magnetosensors were able to detect 0.4 fmol of a synthetic target and endogenous miR-21 (selected as a model for its role in a wide variety of cancers) in only 2 h in total RNA extracted from cancer cells and human breast-tumor specimens without PCR amplification and sample preprocessing. These results open up formidable perspectives for the diagnosis and prognosis of human cancers and for drug-discovery programs.

Fibroblast activation protein expression by stromal cells and tumor-associated macrophages in human breast cancer

Fibroblast activation protein (FAP) has long been known to be expressed in the stroma of breast cancer. However, very little is known if the magnitude of FAP expression within the stroma may have a prognostic value and reflect the heterogeneous biology of the tumor cell. An earlier study had suggested that stromal FAP expression in breast cancer was inversely proportional to prognosis. We, therefore, hypothesized that stromal FAP expression may correlate with clinicopathologic variables and may serve as an adjunct prognostic factor in breast cancer. We evaluated the expression of FAP in a panel of breast cancer tissues (n = 52) using a combination of immunostain analyses at the tissue and single-cell level using freshly frozen or freshly digested human breast tumor samples, respectively. Our results showed that FAP expression was abundantly expressed in the stroma across all breast cancer subtypes without significant correlation with clinicopathologic factors. We further identified a subset of FAP-positive (or FAP(+)) stromal cells that also expressed CD45, a pan-leukocyte marker. Using freshly dissociated human breast tumor specimens (n = 5), we demonstrated that some of these FAP(+)CD45(+) cells were CD11b(+)CD14(+)MHC-II(+), indicating that they were likely tumor-associated macrophages (TAMs). Although FAP(+)CD45(+) cells have been demonstrated in the mouse tumor stroma, our results demonstrating that human breast TAMs expressed FAP were novel and suggested that existing and future FAP-directed therapy may have dual-therapeutic benefits targeting both stromal mesenchymal cells and immune cells such as TAMs. More work is needed to explore the role of FAP as a potential targetable molecule in breast cancer treatment.

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.

MiRNA Gene Promoters Are Frequent Targets of Aberrant DNA Methylation in Human Breast Cancer

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

Interleukin enhancer-binding factor 3 promotes breast tumor progression by regulating sustained urokinase-type plasminogen activator expression

Sustained urokinase-type plasminogen activator (uPA) expression is detected in aggressive breast tumors. Although uPA can be transiently upregulated by diverse extracellular stimuli, sustained, but not transiently-upregulated uPA expression contributes to breast cancer invasion/metastasis. Unfortunately, how sustained uPA expression is achieved in invasive/metastatic breast cancer cells is unknown. Here, we show that sustained and transiently-upregulated uPA expression are regulated by distinct mechanisms. Using a collection of transcription factor-targeted siRNAs, we discovered that interleukin enhancer binding factor 3 (ILF3) is required for sustained uPA expression. Two discrete mechanisms mediate ILF3 action. The first is that ILF3 activates uPA transcription by binding to the CTGTT sequence in the nucleotides -1,004~-1,000 of the uPA promoter; the second is that ILF3 inhibits the processing of uPA mRNA-targeting pri-miRNAs. Knockdown of ILF3 led to significant reduction in in vitro cell growth/migration/invasion and in vivo breast tumor development. Importantly, immunohistochemistry showed that nuclear ILF3, but not cytoplasmic ILF3 staining correlates with elevated uPA level and higher grades of human breast tumor specimens. Nuclear localization of ILF3 highlights the role of ILF3 in sustained uPA expression as a transcription activator and pri-miRNA processing blocker. In conclusion, this study shows that ILF3 promotes breast tumorigenicity by regulating sustained uPA expression.

Mitochondrial genome instability resulting from SUV3 haploinsufficiency leads to tumorigenesis and shortened lifespan

Mitochondrial dysfunction has been a hallmark of cancer. However, whether it plays a causive role awaits to be elucidated. Here, using an animal model derived from inactivation of SUV3, a mitochondrial helicase, we demonstrated that mSuv3+/− mice harbored increased mtDNA mutations and decreased mtDNA copy numbers, leading to tumor development in various sites and shortened lifespan. These phenotypes were transmitted maternally, indicating the etiological role of the mitochondria. Importantly, reduced SUV3 expression was observed in human breast tumor specimens compared to corresponding normal tissues in two independent cohorts. These results demonstrated for the first time that maintaining mtDNA integrity by SUV3 helicase is critical for cancer suppression.

Abstract 916: Role of delta16HER2 splice variant in HER2-driven tumor progression and response to targeted therapy

Abstract We reported that the splice variant of human HER2 lacking exon 16 (delta16HER2) represents a highly penetrating HER2 oncogenic alteration identified in human primary breast tumor specimens and is able to influence the response to Trastuzumab. This HER2 variant forms covalent cysteine bonds that generate constitutively active homodimers, thereby activating multiple oncogenic downstream signaling pathways that we recently found to be mediated through activated Src kinase. To examine the ability of delta16HER2 to transform mammary epithelium in vivo and to monitor delta16HER2-driven tumorigenesis in live mice, we generated a FVB transgenic mouse model for the human delta16HER2 isoform. Transgenic female mice developed multifocal mammary tumors with a rapid onset starting at about 12 weeks of age and progressively thereafter, clearly pointing to the candidacy of the delta16HER2 isoform as the transforming form of the human HER2 oncoprotein. Histological and immunohistochemical analysis (IHC) of primary mammary nodules revealed a population of polygonal cells with classical epithelia-like aspects distinctly expressing HER2 and also a population of smaller spindle-shaped cells arranged in fascicles with lower levels of HER2 expression, suggesting the onset of the epithelial-to-mesenchymal transition (EMT). Consistent with these findings, FACS analysis of delta16HER2-positive tumor cells immunomagnetically purified from disaggregated transgenic primary tumors indicated the increased mean fluorescence intensity of HER2 staining with increasing tumor cell size. IHC analysis of the lung metastases that had formed in the majority of female mice revealed monomorphic and classical epithelial tumor cells homogeneously expressing high levels of delta16HER2. FACS and IHC analyses confirmed the lower binding efficacy of Trastuzumab to delta16HER2-overexpressing primary tumor cells cultured both under bidimensional (2D) and tridimensional (3D) conditions as compared to monoclonal reagents directed to different HER2 extracellular domain epitopes. Experiments in both primary and metastatic in vitro and in vivo delta16HER2-positive models are in progress to determine whether delta16HER2-driven tumor aggressiveness and Trastuzumab susceptibility depend not only on genetic changes intrinsic to the tumor cell, i.e., the EMT process, but also on extrinsic tumor surrounding microenvironment-related factors such as an imbalance between extracellular and intracellular pH, redox state and hypoxia. Preliminary FACS and IHC analyses indicate that delta16HER2-positive primary tumor cells are reactive for known epithelial markers as EpCAM, E-cadherin- and ck-18 and, a small subset of these mammary tumor cells, also stain positive for the mesenchymal differentiation markers vimentin, N-cadherin and ck14 significantly indicating an active EMT program. Supported by AIRC and Ministry of Health Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916. doi:1538-7445.AM2012-916