Tumorigenesis In Human Breast Cancer Research Articles

Reduction of phosphorylated signal transducer and activator of transcription-5 expression in feline mammary carcinoma.

Signal transducers and activators of transcription (STATs) are a family of transcription factors involved in various normal physiological cellular processes. Moreover, STATs have been recently identified as novel therapeutic targets for various human tumors. STAT3, STAT5a, and STAT6 have been suggested to be involved in tumorigenesis in human breast cancer. Owing to the similarity between feline mammary carcinomas (FMCs) and human breast cancers, these factors may play an important role in FMCs. However, studies on the expression of STATs in animal tumors are limited. Therefore, in this study, we aimed to characterize the expression of total STAT5 (tSTAT5) and phosphorylated STAT5 (pSTAT5) in FMCs, feline mammary adenomas, non-neoplastic proliferative mammary gland lesions, and normal feline mammary glands using immunohistochemistry. High expression of tSTAT5 was observed in the cytoplasm of all the samples assessed in this study. Moreover, high expression of tSTAT5 was observed in the nucleus; however, its levels varied depending on the lesion. The percentage of pSTAT5-nuclear positive cells varied among normal feline mammary glands (40.1 ± 25.1%), and non-neoplastic lesions, including mammary hyperplasia (43.2 ± 28.6%) and fibroadenomatous changes (18.0 ± 13.6%). Moreover, the percentage of pSTAT5-nuclear-positive cells in feline mammary adenomas was 24.5 ± 19.2%, which was significantly reduced in feline mammary carcinomas (2.4 ± 5.6%), regardless of histopathological subtype. This study suggests that decreased STAT5 activity may be involved in the development and malignant progression of feline mammary carcinomas.

Expression of acetylated histones H3 and H4 and histone deacetylase enzymes HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas of female dogs.

Histone deacetylation is an important mechanism involved in human breast cancer tumorigenesis and recent veterinary oncology studies also demonstrate a similar relationship in some canine neoplasms. The use of HDAC inhibitors in vitro and in vivo has demonstrated antitumor action on several strains of human and animal cancers. The present study aims to correlate the expression of H3K9Ac, H4K12Ac, HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas in dogs with clinicopathological parameters and overall survival time. To this end, 61 samples of simple breast carcinomas were analyzed by the immunohistochemistry technique with subsequent validation of the antibodies by the Western Blot technique. The expressions obtained via a semi-quantitative way were categorized by assigning scores and classified into high or low expressions according to the given score, except for HDAC6, when the marking percentage was considered and subdivided into high and low expressions using the median value. For statistical analysis, the chi-square test or Fisher exact test were used as univariate analysis and correspondence analysis as a multivariate test, in addition to the Kaplan-Meier survival analysis. In the studied samples, the highest frequencies were determined for the high expression proteins H4K12Ac (88.5%), HDAC2 (65.6%) and HDAC6 (56.7%) and the low expression proteins H3K9Ac (73.8%) and HDAC1 (54.1%). An association between the low expression of HDAC1 and the presence of lymph node metastasis (p = 0.035) was indicated by univariate analysis while the high expression of HDAC1 was associated with favorable prognostic factors, such as the absence of lymph node metastasis and low mitotic index by multivariate analysis. Also, by multivariate analysis, the low expression of HDAC6 was correlated with the low expression of Ki67, smaller tumors, and better prognosis factors as well. Protein expression was not correlated with patients' overall survival time (p > 0.05). The high expressions of HDAC2 and HDAC6 in mammary carcinomas in female dogs may be useful information for research involving therapeutic targets with iHDACs since their inhibition favors hyperacetylation and transcription of tumor suppressor genes.

Integrated Molecular Characterization to Reveal the Association between Kynurenine 3-Monooxygenase Expression and Tumorigenesis in Human Breast Cancers.

Kynurenine 3-monooxygenase (KMO) is overexpressed in several tumors and participates in the progression of breast cancer tumorigenesis, including cancer types such as triple-negative breast cancer (TNBC). This malignant gene is an enzyme in the kynurenine pathway, which is involved in the carcinogenesis of cancer through immune function manipulation. However, it remains unclear whether the role of the KMO contributes to tumorigenesis and immune functions in human breast cancer. In this study, we found that KMO was highly expressed in different types of tumors, especially in invasive ductal breast carcinoma. In addition, KMO expression was positively correlated with the malignant clinical features of patients with breast cancer, such as TNBC and a nodal-positive status, along with patients with a higher Nottingham prognostic index (NPI). Furthermore, the top ten KMO-correlated genes were the chemokines and pro-inflammatory cytokines known to be involved in the progression of various cancers, therefore, KMO may facilitate breast cancers via synergistically regulating inflammatory responses in tumors with these hub genes. Taken together, these findings highlight the tumor-promotion role of KMO in breast cancers and suggest that KMO can serve as a biomarker for prognosis prediction in breast cancer patients.

Abstract 162: DNA methylation activates TP73 expression in human breast cancer tumorigenesis

Abstract Epigenetic events that cause changes in gene expression are common in human cancers. However, the connection between TP73 expression and its epigenetic functionality in tumorigenesis has not been widely explored. As a transcription factor, p73 is activated in a similar manner to p53 in response to DNA damage and regulates the expression of downstream genes involved in cell cycle arrest and apoptosis. However, there are other compounding functions of this gene that reflect its non-tumor-related characters, thus making it very difficult to assess its specific role in tumorigenesis, which results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different proteins TAp73 and ΔNp73 and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancers. Therefore, the role of TP73 in tumorigenesis has remained elusive to date. In this study, we determined TP73 expression in normal human mammary epithelial cells (HMEC), non-cancerous Li-Fraumeni Syndrome (LFS) skin fibroblasts, human breast cancer cell lines (MCF7 & MDA321), neuroblastoma cell lines (IMR32 & SK-N-SH) and normal skin fibroblast cell line (HS27). We also conducted an immunohistochemistry analysis of TP73 expression in human normal breast tissues, cancerous breast tissues and non-cancerous (cancer-adjacent) breast tissues. The results show that TP73 expression is evident only in cancer cell lines and breast cancer tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) and tumor protein p53 (TP53) bind to TP73 promoter and regulate TP73 expression. These observations demonstrate the existence of a positive correlation at the promoter site of TP73 between its expression and DNA methylation. However, TP73 expression is not involved in cell proliferation, which may prove significant for the development of future diagnostic and therapeutic applications. Citation Format: Zhixing Yao, David R. Yao, William Yu, Zaki A. Sherif. DNA methylation activates TP73 expression in human breast cancer tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 162.

MELK as a potential target to control cell proliferation in triple-negative breast cancer MDA-MB-231 cells.

Maternal embryonic leucine zipper kinase (MELK) is an important regulator in tumorigenesis of human breast cancer, and if silenced leads to programmed cell death in specific breast cancer cell lines, including MDA-MB-231 cells. In the present study, RNA interference, proliferation assay and semi-quantification of cell cycle relative proteins were performed to determine the effects of MELK in human breast cancer cells. Data demonstrated that the highest level of MELK protein in the MDA-MB-231 cell line among eight breast cancer cell lines. The sensitivity of MELK small interfering-RNA varied in different breast cancer cell lines, but MELK silencing resulted in marked suppression of proliferation of triple-negative breast cancer (TNBC) and non-TNBC cells. Specific silencing of MELK caused G2 arrest in TNBC MDA-MB-231 and HCC1143 cells, and G1 arrest in non-TNBC T47D and MCF7 cells. Notably, the knockdown of MELK did not induce apoptosis in HCC1143 cells, indicated by the lack of caspase-3 expression. In addition, in response to MELK silencing, cyclin B and cyclin D1 were downregulated in four breast cancer cell lines. Furthermore, the silencing of MELK resulted in the upregulation of p21, p27 and phosphorylated (p)-c-Jun N-terminal kinase (JNK) in HCC1143 TNBC cells, and downregulation of p21 and p-JNK in T47D non-TNBC cells. Additionally, MELK protein was markedly suppressed in non-TNBC cells in response to estrogen deprivation. The findings from the present study suggested that MELK may be a potential target in MDA-MB-231 cells, although genetic knockdown of MELK resulted in inhibitory effects on proliferation of TNBC and non-TNBC cells. MELK exert its effect on different breast cancer cells via arrest of different cell cycle phases and therefore mediated by different mediators, which may be involved in the crosstalk with MELK signaling and with the estrogen receptor signaling pathway.

Bufalin inhibits human breast cancer tumorigenesis by inducing cell death through the ROS-mediated RIP1/RIP3/PARP-1 pathways.

Bufalin, a key active ingredient of the Chinese medicine Chan Su, inhibits breast cancer tumorigenesis in vitro and in vivo. Here, we found that the pan-caspase inhibitor zVAD-fmk failed to inhibit bufalin-induced cell death in MCF-7 and MDA-MB-231 human breast cancer cells, confirming that the cell death induced by bufalin is caspase-independent. Instead, bufalin increased the expression of the necroptosis mediators RIP1 and RIP3. Bufalin-induced cell death was prevented by small molecule inhibitors of RIP1 and poly (ADP-ribose) polymerase-1 (PARP-1) or genetic knockdown of RIP3 by shRNA transfection. In addition, ectopic RIP3 expression enhanced cell death by bufalin. We also found that bufalin increased intracellular reactive oxygen species levels; and cell death by bufalin was inhibited by the antioxidant NAC. In a mouse xenograft model of human breast cancer, bufalin induced PARP-1-dependent tumor cell death and inhibited tumor growth. These results demonstrated that bufalin inhibits human breast cancer tumorigenesis by inducing cell death through the reactive oxygen species-mediated RIP1/RIP3/PARP-1 pathways.

MiR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

ABSTRACTDrosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

MiR-19b suppresses PTPRG to promote breast tumorigenesis.

Protein tyrosine phosphatase receptor type G (PTPRG) is an important tumor suppressor gene in multiple human cancers. In this study, we found that PTPRG protein levels were downregulated in breast cancer tissues while the mRNA levels varied irregularly, implying a post-transcriptional mechanism was involved. Because microRNAs are powerful post-transcriptional regulators of gene expression, we used bioinformatics analysis to search for microRNAs that potentially targets PTPRG in the setting of breast cancer. We identified two specific binding sites for miR-19b in the 3′-untranslated region of PTPRG. We further identified an inverse correlation between miR-19b and PTPRG protein levels, but not mRNA levels, in human breast cancer tissues. By overexpressing or knocking down miR-19b in MCF-7 cells and MDA-231 cells, we experimentally confirmed that miR-19b directly suppresses PTPRG expression. Furthermore, we determined that the inhibition of PTPRG by miR-19b leads to increased proliferation, stimulated cell migration and reduced apoptosis. Taken together, our findings provide the first evidence that miR-19b inhibits PTPRG expression to promote tumorigenesis in human breast cancer.

METCAM/MUC18 promoted tumorigenesis of human breast cancer SK-BR-3 cells in a dosage-specific manner

ObjectiveOverexpression of METCAM/MUC18, an immunoglobulin-like cell-adhesion molecule, promotes tumorigenesis and progression of human breast cancer cells. We also observed an intriguing phenomenon that a high-expressing SK-BR-3 clone manifested a transient tumor suppression effect in vivo. The purpose of this study was to understand if this was caused by clonal variation, METCAM/MUC18-dosage effect, or the number of cells injected. Materials and MethodsSeveral G418-resistant clones of SK-BR-3, expressing different levels of METCAM/MUC18, were obtained for testing effects of human METCAM/MUC18 on in vitro motility, invasiveness, and anchorage-independent colony formation (in vitro tumorigenicity) and in vivo tumorigenesis in female Balb/C athymic nude mice. Tumor sections were made for histology and immunohistochemistry analyses, and tumor lysates for Western blot analysis to determine the effects of human METCAM/MUC18 expression on levels of various downstream effectors. ResultsMETCAM/MUC18 promoted in vitro motility, invasiveness, and in vitro tumorigenicity of SK-BR-3 cells in a dosage-specific manner. Overexpression of METCAM/MUC18 could promote in vivo tumorigenesis of SK-BR-3 cells even when one tenth of the previously used cell number (5 × 105) was injected and in vivo tumorigenesis of SK-BR-3 cells was directly proportional to the dosage of the protein. The previously observed transient tumor suppression effect from the same clone was no longer observed. The downstream effector, such as phospho-AKT/AKT ratio, was elevated in the tumors. ConclusionTransient suppression observed previously in the clone was caused by injection of a high cell number (2 × 106–5 × 106). METCAM/MUC18 positively promotes tumorigenesis of SK-BR-3 cells by increasing the survival and proliferation pathway.

Reduced expression of sushi domain containing 2 is associated with progression of non-small cell lung cancer.

Sushi domain containing 2 (SUSD2) has been identified as a gene encoding an 822-amino acid protein, which contains a transmembrane domain and functional domains inherent to adhesion molecules. Previous studies have reported that increased expression of SUSD2 has a critical role in tumorigenesis in human breast cancer. However, to the best of our knowledge, SUSD2 expression status and its correlation with the clinicopathological features of non-small cell lung cancer (NSCLC) have not previously been investigated. In the present study, reverse transcription-quantitative polymerase chain reaction and western blotting were used to evaluate SUSD2 messenger RNA (mRNA) and protein expression in NSCLC and adjacent normal lung tissues. The clinicopathological significance of SUSD2 was investigated by immunohistochemical analysis of an NSCLC tissue microarray. Receiver operating characteristic analysis was used to determine the cut-off score for positive expression of SUSD2. Furthermore, the correlation between SUSD2 expression and the clinicopathological features of NSCLC was analyzed by χ2 test. The results revealed that SUSD2 mRNA (P<0.0001) and protein (P<0.0001) expression levels were significantly decreased in NSCLC tissues compared with those of adjacent normal tissues. When the SUSD2 positive expression percentage was determined to be >47.5% (area under ROC curve, 0.799; P<0.000), positive expression of SUSD2 was observed in 100% (32/32) of normal lung tissues and 55% (88/160) of NSCLC tissues by immunohistochemistry (χ2=21.160; P<0.000). Furthermore, it was demonstrated that the reduced SUSD2 protein levels in cancer tissues were positively correlated with poor histological grade (χ2=41.764; P<0.000), advanced clinical stage (χ2=10.790; P=0.013), higher pT (χ2=9.070; P=0.028) and positive regional lymph node metastasis (χ2=15.399; P=0.002). In conclusion, these data suggest that the reduced expression of SUSD2 is associated with the progression of NSCLC and may have a role in the pathogenesis of NSCLC.

Abstract 1445: Overexpression of angiotensin II type 1 receptor induces epithelial-mesenchymal transition and promotes tumorigenesis of human breast cancer cells

Abstract Propose: Accumulating evidence suggests that angiotensin II type 1 receptor (AGTR1) is involved in tumorigenesis and metastasis in many types of cancer, however little is known about its role in breast cancer. Our objective of the present study is to investigate the effect of AGTR1 overexpression on cell proliferation, epithelial-mesenchymal transition (EMT), migration, invasion, angiogenesis and tumorigenesis of human breast cancer. Methods: To determine the role of AGTR1, MCF7 cells were stably transfected with an expression construct carrying the GFP cDNA for AGTR1. MTS assay, Matrigel invasion assay, wound healing assay, Western blotting and immunofluorescence were used. For in vivo study, parental MCF7 or AGTR1-MCF7 cells were subcutaneously injected into the left and right flanks of nude mice. Tumor growth and angiogenesis were determined. Results and conclusion: AGTR1-MCF7 cells, expressing high level of AGTR1 exhibited significant increases in cell proliferation, migration and invasion capacities compared with parental MCF7 cells. Furthermore, AGTR1-MCF7 cells displayed the mesenchymal phenotype, as evidenced by elongated spindle morphology, decreased E-cadherin level, increased N-cadherin and snail, and nuclear accumulation of smad3 and smad4. Interestingly, blockade of smad4 using small interfering RNA strongly induced mesenchymal to epithelial transition (MET) and restoration of E-cadherin expression in AGTR1-MCF7 cells, suggesting that AGTR1-induced EMT is ultimately mediated by smad4 activation. Finally, we showed that AGTR1 overexpression enhanced tumor volume (p&amp;lt;0.001, n = 10) and weight (p&amp;lt;0.01) as well as increased angiogenesis in human breast cancer xenograft in vivo. In summary, we demonstrate that AGTR1 overexpression promotes cell proliferation, EMT, angiogenesis and tumorigenic properties of breast cancer cells. Our findings suggest that AGTR1 could be a useful target of therapy in breast cancer. Citation Format: Eunhye Oh, Ji Young Kim, Youngkwan Cho, Nahyun Lee, Hyunsook An, Jun Suk Kim, Jae Hong Seo. Overexpression of angiotensin II type 1 receptor induces epithelial-mesenchymal transition and promotes tumorigenesis of human breast cancer cells. [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 1445. doi:10.1158/1538-7445.AM2015-1445

Cyclin-dependent kinase 11(p110) (CDK11(p110)) is crucial for human breast cancer cell proliferation and growth.

Cyclin-dependent kinases (CDKs) play important roles in the development of many types of cancers by binding with their paired cyclins. However, the function of CDK11 larger protein isomer, CDK11p110, in the tumorigenesis of human breast cancer remains unclear. In the present study, we explored the effects and molecular mechanisms of CDK11p110 in the proliferation and growth of breast cancer cells by determining the expression of CDK11p110 in breast tumor tissues and examining the phenotypic changes of breast cancer cells after CDK11p110 knockdown. We found that CDK11p110 was highly expressed in breast tumor tissues and cell lines. Tissue microarray analysis showed that elevated CDK11p110 expression in breast cancer tissues significantly correlated with poor differentiation, and was also associated with advanced TNM stage and poor clinical prognosis for breast cancer patients. In vitro knockdown of CDK11p110 by siRNA significantly inhibited cell growth and migration, and dramatically induced apoptosis in breast cancer cells. Flow cytometry demonstrated that cells were markedly arrested in G1 phase of the cell cycle after CDK11p110 downregulation. These findings suggest that CDK11p110 is critical for the proliferation and growth of breast cancer cells, which highlights CDK11p110 may be a promising therapeutic target for the treatment of breast cancer.

Lentivirus mediated silencing of ubiquitin specific peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro.

BackgroundUbiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells.ResultsIn the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.ConclusionsAll our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0006-y) contains supplementary material, which is available to authorized users.

WNT-1 inducible signaling pathway protein-1 enhances growth and tumorigenesis in human breast cancer.

WNT1 inducible signaling pathway protein 1 (WISP1) plays a key role in many cellular functions in a highly tissue-specific manner; however the role of WISP1 in breast cancer is still poorly understood. Here, we demonstrate that WISP1 acts as an oncogene in human breast cancer. We demonstrated that human breast cancer tissues had higher WISP1 mRNA expression than normal breast tissues and that treatment of recombinant WISP1 enhanced breast cancer cell proliferation. Further, ectopic expression of WISP1 increased the growth of breast cancer cells in vitro and in vivo. WISP1 transfection also induced epithelial-mesenchymal-transition (EMT) in MCF-7 cells, leading to higher migration and invasion. During this EMT-inducing process, E-cadherin was repressed and N-cadherin, snail, and β-catenin were upregulated. Filamentous actin (F-actin) remodeling and polarization were also observed after WISP1 transfection into MCF-7 cells. Moreover, forced overexpression of WISP1 blocked the expression of NDRG1, a breast cancer tumor suppressor gene. Our study provides novel evidence that WISP1-modulated NDRG1 gene expression is dependent on a DNA fragment (−128 to +46) located within the human NDRG1 promoter. Thus, we concluded that WISP1 is a human breast cancer oncogene and is a potential therapeutic target.

Acylglycerol kinase promotes cell proliferation and tumorigenicity in breast cancer via suppression of the FOXO1 transcription factor.

BackgroundAcylglycerol kinase (AGK) is reported to be overexpressed in multiple cancers. The clinical significance and biological role of AGK in breast cancer, however, remain to be established.MethodsAGK expression in breast cancer cell lines, paired patient tissues were determined using immunoblotting and Real-time PCR. 203 human breast cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the relationship between AGK expression and the clinicopathological features of breast cancer. Functional assays, such as colony formation, anchorage-independent growth and BrdU assay, and a xenograft tumor model were used to determine the oncogenic role of AGK in human breast cancer progression. The effect of AGK on FOXO1 transactivity was further investigated using the luciferase reporter assays, and by detection of the FOXO1 downstream genes.ResultsHerein, we report that AGK was markedly overexpressed in breast cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of AGK significantly correlated with patients’ clinicopathologic characteristics, including clinical stage and tumor-nodule-metastasis (TNM) classification. Breast cancer patients with higher levels of AGK expression had shorter overall survival compared to patients with lower AGK levels. We gained valuable insights into the mechanism of AGK expression in breast cancer cells by demonstrating that overexpressing AGK significantly enhanced, whereas silencing endogenous AGK inhibited, the proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo. Furthermore, overexpression of AGK enhanced G1-S phase transition in breast cancer cells, which was associated with activation of AKT, suppression of FOXO1 transactivity, downregulation of cyclin-dependent kinase inhibitors p21 Cip1 and p27 Kip1 and upregulation of the cell cycle regulator cyclin D1.ConclusionsTaken together, these findings provide new evidence that AGK plays an important role in promoting proliferation and tumorigenesis in human breast cancer and may serve as a novel prognostic biomarker and therapeutic target in this disease.

Abstract P2-05-03: Role of tumor immune environment in tumor initiation and growth rates in mouse mammary tumor models

Abstract The role of the tumor immune environment is critical in human breast cancer tumorigenesis and response to therapy: tumors with a Th1 immune activating environment (high cytotoxic CD8+ T cells and low FoxP3 and myeloid derived suppressor cells (MDSC)) show improved response to chemotherapy and improved disease free survival and prognosis, where tumors with a Th2 immune suppressive environment (increased FoxP3, increased MDSC, and decreased CD8 T cells) shows poor response to chemotherapy and worse disease free survival and prognosis. There has not been good correlation with animal models of mammary tumorigenesis and human response to chemotherapy because in mouse models (1) tumor implant models do not show the tumor immune infiltration seen in human tumors (2) xenograph models are not immunocompetent (3) the tumor immune infiltration has not been evaluated the spontaneous mouse mammary models. We evaluated the tumor immune environment in relation to time to tumor development and rate of tumor growth in two commonly used transgenic mouse models: TgMMTV-neu and C3(1)Tag to better define role of the tumor immune environment in tumorigenesis in spontaneous mouse mammary tumor models. Spontaneous tumorigenesis was studied in 80 TgMMTV-neu and 58 C3(1)Tag mice who were observed for tumor development from 6 weeks until tumor ∼1000 mm3. Differences in (1) time to tumor development (2) rate of tumor growth (3) tumor immune environment were evaluated. Between the transgenic mouse models, TgMMTV-neu mice (n = 10) developed tumors later, around 35.0 weeks, and the tumors grew slower with an average growth rate of 47.9 mm3/week than C3(1)Tag mice which developed tumors around 18.2 weeks with an average growth rate of 88.1 (p = 0.006). The tumor infiltration of MDSC inhibitory cells were higher in the C3(1)Tag mice than the TgMMTV-neu mice and approaches significance (17.4% of all lymphocytes in C3(1)Tag and 4.1% in TgMMTV-neu p = 0.08). Furthermore, there are increased CD8+ T cells in TgMMTV-neu mouse tumors than in C3(1)Tag (34.08% CD3+ cells in TgMMTV-neu mice and 22.38% in C3(1)Tag mice, p = 0.07) with similar FoxP3 levels (6.4% in TgMMTV-neu and 10.2% in C3(1)Tag mice p = 0.2) therefore the CD8/FoxP3 ratio in TgMMTV-neu mice is 5.3 where the CD8/FoxP3 ratio in C3(1)Tag mice is 2.2. These data demonstrate that C3(1)Tag mice have more aggressive tumorigenesis than the TgMMTV-neu mice and a more immunosuppressive tumor environment (higher MDSC, lower CD8, and smaller CD8/FoxP3 ratio) supporting the tumor immune environment plays an important role in tumorigenesis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-03.

Knockdown of FLOT1 Impairs Cell Proliferation and Tumorigenicity in Breast Cancer through Upregulation of FOXO3a

Lipid rafts, specialized domains in cell membranes, function as physical platforms for various molecules to coordinate a variety of signal transduction processes. Flotinllin-1 (FLOT1), a marker of lipid rafts, is involved in the progression of cancer, but the precise mechanism remains unclear. The aim of the present study was to examine the role of FLOT1 on the tumorigenesis of breast cancer cells and its clinical significance in progression of the disease. FLOT1 expression was analyzed in 212 paraffin-embedded, archived clinical breast cancer samples by using immunohistochemistry (IHC). The effect of FLOT1 on cell proliferation and tumorigenesis was examined in vitro and in vivo. Western blotting and luciferase reporter analyses were carried out to identify the effects of downregulating FLOT1 on expression of cell cycle regulators and transcriptional activity of FOXO3a. IHC analysis revealed high expression of FLOT1 in 129 of the 212 (60.8%) paraffin-embedded archived breast cancer specimens. The overall expression level of FLOT1 significantly correlated with clinical staging and poor patient survival of breast cancer. Strikingly, we found that silencing FLOT1 inhibited proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo, which was further shown to be mechanistically associated with suppression of Akt activity, enhanced transcriptional activity of FOXO3a, upregulation of cyclin-dependent kinase inhibitor p21(Cip1) and p27(Kip1), and downregulation of the CDK regulator cyclin D1. FLOT1 plays an important role in promoting proliferation and tumorigenesis of human breast cancer and may represent a novel prognostic biomarker and therapeutic target for the disease.

SATB1: a 'master genome organizer' in mammary carcinogenesis.

Abstract Abstract #3070 Background: SATB1 is a nuclear protein that has been recently reported to be a 'genome organizer' that delinates specific epigenetic modifications at target gene loci, directly up-regulating metastasis-associated genes while down-regulating tumor-suppressor genes. This study aimed to examine the level of mRNA expression of SATB1 gene in normal and malignant breast tissue and correlate the level to the clinico-pathological parameters.&amp;#x2028; Material and Methods: One hundred and fifteen breast cancer tissue and 31 normal breast tissues were analyzed. Levels of transcription of SATB1 were determined using real-time quantitative PCR and were analyzed against TNM stage, nodal involvement, tumor grade and clinical outcome.&amp;#x2028; Results: The levels of SATB1 mRNA were significantly higher in malignant compared with normal breast tissue (p=0.0167). The expression of SATB1 mRNA was demonstrated to increase with increasing TNM stage (TNM1 vs. TNM2, p=0.0264; TNM2 vs. TNM3, p=0.024) and increasing tumor grade (grade 1 vs. grade 3, p=0.017; grade 2 vs. grade 3, p=0.0437; grade 1 vs. grade 2&amp;3, p=0.021). After a median follow up of 10 years, there was a trend for tumors with higher SATB1 expression levels to be associated with shorter overall survival times.&amp;#x2028; Discussion: These results are consistent with the notion that SATB1 acts as the 'master genome organizer' in tumorigenesis of human breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3070.

Metastasis Tumor Antigen Family Proteins during Breast Cancer Progression and Metastasis in a Reliable Mouse Model for Human Breast Cancer

Chromatin remodeling pathways are critical in the regulation of cancer-related genes and are currently being explored as potential targets for therapeutic intervention. The metastasis tumor antigen (MTA) family of proteins, MTA1, MTA2, and MTA3, are components of chromatin remodeling pathways with potential roles in breast cancer. Although all three MTA family proteins have been shown to be associated with metastatic progression of breast cancers, the expression characteristic of MTA1-3 proteins in a multistep breast cancer progression model remains unknown. Structural and functional studies have suggested that they are heterogeneous in the Mi-2/NuRD complex, exhibit tissue-specific patterns of expression, and impart unique properties to estrogen receptor-alpha (ERalpha) action. This led us to hypothesize that each member of the MTA family possesses a unique role and interacts with different pathways in the stepwise process of breast cancer development and progression. MTA family proteins were examined by immunohistochemistry in breast cancer processes ranging from normal duct, to premalignant lesions, to invasive carcinoma, and to metastasized tumors in PyV-mT transgenic mice, which represents a reliable model for multistage tumorigenesis of human breast cancer. We also determined the association of MTA proteins with the status of cell proliferation, ER, E-cadherin and cytoplasmic beta-catenin, and cancer-related coactivators, AIB1 and PELP1. The expression of all three MTA proteins was altered in primary breast tumors. Each MTA protein had a unique expression pattern during the primary breast tumor progression. Altered expression of MTA1 was observed in both premalignant lesion and malignant carcinoma, but an elevated nuclear expression was observed in ER-negative carcinomas. MTA3 was exclusively expressed in a subset of cells of ER-positive premalignant lesions but not in carcinomas. MTA2 expression seems to be unrelated to ER status. Loss of MTA3 expression and more nuclear localization of MTA1 occurred with loss of E-cadherin and decreased cytoplasmic beta-catenin, two molecules essential for epithelial cell adhesion and important tumor cell invasion. At the late stage of tumor formation, MTA1 is usually expressed in the center of tumors. Coincidentally, the distribution of MTA1-positive cells at this stage was complementary to that of AIB1 and PELP1, which were localized to the tumor periphery with relatively active cell proliferation, scattered ER-positive cells and a limited differentiation. In metastasized lung tumors, the expression pattern of MTA-protein expression was distinct from that in primary counterparts. The findings presented here support the notion that each member of the MTA family might potentially play a stepwise role in a cell type-specific manner during breast cancer progression to metastasis. On the basis of the noted temporal expression patterns of MTA proteins with ER status, cell adhesion-essential regulators (E-cadherin and cytoplasmic beta-catenin), and coactivators, we propose that MTA protein-related chromatin remodeling pathways interact with steroid receptors, growth factor receptors, and other transcriptional signaling pathways to orchestrate the governing of events in breast cancer progression and metastasis.