Malignant Progression Of Breast Cancer Research Articles

Acetylation targets HSD17B4 for degradation via the CMA pathway in response to estrone

ABSTRACTDysregulation of hormone metabolism is implicated in human breast cancer. 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4) catalyzes the conversion of estradiol (E2) to estrone (E1), and is associated with the pathogenesis and development of various cancers. Here we show that E1 upregulates HSD17B4 acetylation at lysine 669 (K669) and thereby promotes HSD17B4 degradation via chaperone-mediated autophagy (CMA), while a single mutation at K669 reverses the degradation and confers migratory and invasive properties to MCF7 cells upon E1 treatment. CREBBP and SIRT3 dynamically control K669 acetylation level of HSD17B4 in response to E1. More importantly, K669 acetylation is inversely correlated with HSD17B4 in human breast cancer tissues. Our study reveals a crosstalk between acetylation and CMA degradation in HSD17B4 regulation, and a critical role of the regulation in the malignant progression of breast cancer.

Transcriptomic dynamics of breast cancer progression in the MMTV-PyMT mouse model

BackgroundMalignant breast cancer with complex molecular mechanisms of progression and metastasis remains a leading cause of death in women. To improve diagnosis and drug development, it is critical to identify panels of genes and molecular pathways involved in tumor progression and malignant transition. Using the PyMT mouse, a genetically engineered mouse model that has been widely used to study human breast cancer, we profiled and analyzed gene expression from four distinct stages of tumor progression (hyperplasia, adenoma/MIN, early carcinoma and late carcinoma) during which malignant transition occurs.ResultsWe found remarkable expression similarity among the four stages, meaning genes altered in the later stages showed trace in the beginning of tumor progression. We identified a large number of differentially expressed genes in PyMT samples of all stages compared with normal mammary glands, enriched in cancer-related pathways. Using co-expression networks, we found panels of genes as signature modules with some hub genes that predict metastatic risk. Time-course analysis revealed genes with expression transition when shifting to malignant stages. These may provide additional insight into the molecular mechanisms beyond pathways.ConclusionsThus, in this study, our various analyses with the PyMT mouse model shed new light on transcriptomic dynamics during breast cancer malignant progression.

Long-term exposure of MCF-7 breast cancer cells to ethanol stimulates oncogenic features.

Alcohol consumption is a risk factor for breast cancer. Little is known regarding the mechanism, although it is assumed that acetaldehyde or estrogen mediated pathways play a role. We previously showed that long-term exposure to 2.5 mM ethanol (blood alcohol ~0.012%) of MCF-12A, a human normal epithelial breast cell line, induced epithelial mesenchymal transition (EMT) and oncogenic transformation. In this study, we investigated in the human breast cancer cell line MCF-7, whether a similar exposure to ethanol at concentrations ranging up to peak blood levels in heavy drinkers would increase malignant progression. Short-term (1-week) incubation to ethanol at as low as 1–5 mM (corresponding to blood alcohol concentration of ~0.0048–0.024%) upregulated the stem cell related proteins Oct4 and Nanog, but they were reduced after exposure at 25 mM. Long-term (4-week) exposure to 25 mM ethanol upregulated the Oct4 and Nanog proteins, as well as the malignancy marker Ceacam6. DNA microarray analysis in cells exposed for 1 week showed upregulated expression of metallothionein genes, particularly MT1X. Long-term exposure upregulated expression of some malignancy related genes (STEAP4, SERPINA3, SAMD9, GDF15, KRT15, ITGB6, TP63, and PGR, as well as the CEACAM, interferon related, and HLA gene families). Some of these findings were validated by RT-PCR. A similar treatment also modulated numerous microRNAs (miRs) including one regulator of Oct4 as well as miRs involved in oncogenesis and/or malignancy, with only a few estrogen-induced miRs. Long-term 25 mM ethanol also induced a 5.6-fold upregulation of anchorage-independent growth, an indicator of malignant-like features. Exposure to acetaldehyde resulted in little or no effect comparable to that of ethanol. The previously shown alcohol induction of oncogenic transformation of normal breast cells is now complemented by the current results suggesting alcohol's potential involvement in malignant progression of breast cancer.

Abstract 175: Transcriptomic and epigenetic dynamics of breast cancer progression in the MMTV-PyMT mouse model

Abstract Using RNA-sequencing and ERRBS (enhanced reduced representation bisulphite sequencing), we identified genes involved in breast cancer malignant progression. Human breast cancers have complex molecular mechanisms of progression and metastasis. To improve diagnosis and drug development, it is critical to identify the genes and molecular pathways involved in tumor progression and malignant transition. Using the polyoma middle T antigen (PyMT) mouse model, we profiled gene expression from four different stages of tumor progression, including hyperplasia, adenoma/MIN (mammary intraepithelial neoplasia), early and late carcinoma. We identified a large number of differentially expressed genes in tumors compared with normal mammary gland, enriched in E2F targets and G2M checkpoint genes. Using the temporal information, we found Myc, Sox4 and Casp9 were associated with tumor progression. Using co-expression network analysis, we identified signature modules of panels of genes, revealing novel insights into molecular mechanisms of breast tumorigenesis. For example, Mcm6 and Ncapd2, hub genes in a DNA replication and cell cycle module, predict metastasis in human samples. We also found the Hells, Hmgb2 and Cit genes to be increased in expression in both mouse and human TCGA (The Cancer Genome Atlas) datasets. We complemented these studies by profiling DNA methylation using ERRBS in the same samples. We identified a large number of differentially methylated cytosines (DMCs), enriched in cis-regulatory elements as well as near genes in cancer-related pathways. A small subset of the DMCs was well conserved between the progressive stages of tumorigenesis, with the greatest shift occurring with the transition to malignancy. We also observed a global hypomethylation and a shift towards a subset of loci with hypermethylation with tumor progression. Expression of some tricarboxylic acid (TCA) cycle genes was negatively correlated with DNA methylation, which may reflect altered metabolic regulation in carcinoma. We then constructed a co-methylation network and discovered a module with decreased DNA methylation in tumor that was enriched with genes from WNT and MAPK signaling pathways. In addition, we compared our results to the human TCGA breast carcinoma dataset and found the epigenetic modules to be overall distinctive between the two species. Altogether, our gene expression, DNA methylation and network analyses with the PyMT mouse model shed new light on transcriptional and epigenetic dynamics during breast cancer malignant progression and reveal gene modules that may regulate this biological process. Citation Format: Ying Cai, Ruben Nogales-Cadenas, Quanwei Zhang, Jhih-rong Lin, Wen Zhang, Zhengdong Zhang. Transcriptomic and epigenetic dynamics of breast cancer progression in the MMTV-PyMT mouse model. [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 175.

The cellular distribution of Na+/H+ exchanger regulatory factor 1 is determined by the PDZ-I domain and regulates the malignant progression of breast cancer.

The oncogenic role of ectopic expression of Na+/H+ exchanger regulatory factor 1 (NHERF1) was recently suggested. Here, we show that NHERF1 was upregulated in high grades compared with low grades. Increased NHERF1 expression was correlated with poor prognosis and poor survival. NHERF1 expression was higher in the nucleus of cancer cells than in contiguous non- mammary epithelial cells. A novel mutation, namely NHERF1 Y24S, was identified in human breast cancer tissues and shown to correspond to a conserved residue in the PDZ-I domain of NHERF1. Truncation and mutation of the PDZ-I domain of NHERF1 increased the nuclear distribution of the NHERF1 protein, and this redistribution was associated with the malignant phenotype of breast cancer cells, including growth, migration, and adhesion. The present results suggest a role for NHERF1 in the progression of breast cancer mediated by the nuclear distribution of the NHERF1 protein, as determined by the truncation or key site mutation of the PDZ-I domain.

PLC-β2 is modulated by low oxygen availability in breast tumor cells and plays a phenotype dependent role in their hypoxia-related malignant potential.

Limited oxygen availability plays a critical role in the malignant progression of breast cancer by orchestrating a complex modulation of the gene transcription largely dependent on the tumor phenotype. Invasive breast tumors belonging to different molecular subtypes are characterized by over-expression of PLC-β2, whose amount positively correlates with the malignant evolution of breast neoplasia and supports the invasive potential of breast tumor cells. Here we report that hypoxia modulates the expression of PLC-β2 in breast tumor cells in a phenotype-related manner, since a decrease of the protein was observed in the BT-474 and MCF7 cell lines while an increase was revealed in MDA-MB-231 cells as a consequence of low oxygen availability. Under hypoxia, the down-modulation of PLC-β2 was mainly correlated with the decrease of the EMT marker E-cadherin in the BT-474 cells and with the up-regulation of the stem cell marker CD133 in MCF7 cells. The increase of PLC-β2 induced by low oxygen in MDA-MB-231 cells supports the hypoxia-related reorganization of actin cytoskeleton and sustains invasion capability. In all examined cell lines, but with an opposite role in the ER-positive and ER-negative cells, PLC-β2 was involved in the hypoxia-induced increase of HIF-1α, known to affect both EMT and CD133 expression. Our data include PLC-β2 in the complex and interconnected signaling pathways induced by low oxygen availability in breast tumor cells and suggest that the forced modulation of PLC-β2 programmed on the basis of tumor phenotype may prevent the malignant progression of breast neoplasia as a consequence of intra-tumoral hypoxia. © 2016 Wiley Periodicals, Inc.

Co-expression of parathyroid hormone related protein and TGF-beta in breast cancer predicts poor survival outcome

BackgroundBetter methods to predict prognosis can play a supplementary role in administering individualized treatment for breast cancer patients. Altered expressions of PTHrP and TGF-β have been observed in various types of human cancers. The objective of the current study was to evaluate the association of PTHrP and TGF-β level with the clinicopathological features of the breast cancer patients.MethodsImmunohistochemistry was used to examine PTHrP and TGF-β protein expression in 497 cases of early breast cancer, and Kaplan-Meier method and COX’s Proportional Hazard Model were applied to the prognostic value of PTHrP and TGF-β expression.ResultsBoth over-expressed TGF-β and PTHrP were correlated with the tumor in larger size, higher proportion of axillary lymph node metastasis and later clinical stage. Additionally, the tumors with a high TGF-β level developed poor differentiation, and only TGF-β expression was associated with disease-free survival (DFS) of the breast cancer patients. Followed up for a median of 48 months, it was found that only the patients with negative TGF-β expression had longer DFS (P < 0.05, log-rank test). Nevertheless, those with higher PTHrP expression tended to show a higher rate of bone metastasis (67.6 % vs. 45.8 %, P = 0.019). In ER negative subgroup, those who developed PTHrP positive expression presented poor prognosis (P < 0.05, log-rank test). The patients with both positive TGF-β and PTHrP expression were significantly associated with the high risk of metastases. As indicated by Cox’s regression analysis, TGF-β expression and the high proportion of axillary lymph node metastasis served as significant independent predictors for breast cancer recurrence.ConclusionsTGF-β and PTHrP were confirmed to be involved in regulating the malignant progression in breast cancer, and PTHrP expression, to be associated with bone metastasis as a potential prognostic marker in ER negative breast cancer.

Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway.

Aberrant glycosylation is frequently observed in cancers. Core 1 β1,3-galactosyltransferase (C1GALT1) is an exclusive enzyme in humans that catalyzes the biosynthesis of core 1 O-glycan structure, Gal-GalNAc-O-Ser/Thr, whose expression is commonly up-regulated during tumorigenesis. Little is known about the function of C1GALT1 in breast cancer. This study aims to determine the correlation between C1GALT1 expression and breast cancer clinicopathological features and roles of C1GALT1 in breast cancer malignant phenotypes. Public databases and our data showed that C1GALT1 mRNA and C1GALT1 protein are frequently up-regulated in breast cancer; and increased C1GALT1 expression correlates with higher histological grade and advanced tumor stage. Overexpression of C1GALT1 enhanced breast cancer cell growth, migration, and invasion in vitro as well as tumor growth in vivo. Conversely, C1GALT1 knockdown suppressed these malignant phenotypes. Furthermore, C1GALT1 modulates O-glycan structures on Mucin (MUC) 1 and promotes MUC1-C/β-catenin signaling in breast cancer cells. These findings suggest that C1GALT1 enhances breast cancer malignant progression through promoting MUC1-C/β-catenin signaling pathway. Unveiling the function of C1GALT1 in breast cancer opens new insights to the roles of C1GALT1 and O-glycosylation in tumorigenesis and renders the potential of C1GALT1 as a target of novel therapeutic agent development.

Bone marrow stromal antigen 2 expressed in cancer cells promotes mammary tumor growth and metastasis.

IntroductionSeveral innate immunity genes are overexpressed in human cancers and their roles remain controversial. Bone marrow stromal antigen 2 (BST-2) is one such gene whose role in cancer is not clear. BST-2 is a unique innate immunity gene with both antiviral and pro-tumor functions and therefore can serve as a paradigm for understanding the roles of other innate immunity genes in cancers.MethodsMeta-analysis of tumors from breast cancer patients obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets were evaluated for levels of BST-2 expression and for tumor aggressiveness. In vivo, we examined the effect of knockdown of BST-2 in two different murine carcinoma cells on tumor growth, metastasis, and survival. In vitro, we assessed the effect of carcinoma cell BST-2 knockdown and/or overexpression on adhesion, anchorage-independent growth, migration, and invasion.ResultsBST-2 in breast tumors and mammary cancer cells is a strong predictor of tumor size, tumor aggressiveness, and host survival. In humans, BST-2 mRNA is elevated in metastatic and invasive breast tumors. In mice, orthotopic implantation of mammary tumor cells lacking BST-2 increased tumor latency, decreased primary tumor growth, reduced metastases to distal organs, and prolonged host survival. Furthermore, we found that the cellular basis for the role of BST-2 in promoting tumorigenesis include BST-2-directed enhancement in cancer cell adhesion, anchorage-independency, migration, and invasion.ConclusionsBST-2 contributes to the emergence of neoplasia and malignant progression of breast cancer. Thus, BST-2 may (1) serve as a biomarker for aggressive breast cancers, and (2) be a novel target for breast cancer therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0493-8) contains supplementary material, which is available to authorized users.

Abstract 3150: Exploring the role of Rap1Gap in the progression from DCIS to invasive breast carcinoma

Abstract Understanding the underlying mechanisms that drive malignant progression of breast cancer is important. The purpose of this study is to define the role of Rap1Gap in the progression of ductal carcinoma in-situ (DCIS) to invasive cancer. We employed an in-vitro three-dimensional (3D) overlay model that provides a physiologically relevant microenvironment to study mechanisms of mammary gland development and malignant progression. We previously employed next generation sequencing (NGS) and identified 63 consistently upregulated genes in three DCIS lines (SUM102, SUM225, MCF10.DCIS) compared to non-transformed mammary epithelial cells. To gain insight into common transcriptional elements of co-regulated genes that may be critical to the DCIS phenotype, we have mined our NGS data using Frameworker (Genomatix). The 63 upregulated genes are associated with 244 candidate promoters out of a total of 82,703 promoters in the entire human genome. Our analysis shows that the common framework RXRF-ZF02-ZF02-PLAG-HDBP is only present in the promoters of three genes: RAP1GAP, SPRY4 and PDGFB. All of these genes are upregulated in our DCIS signature, which means that this framework is hugely enriched (336-fold) over what would be expected by chance. Rap1Gap has tumor suppressor properties; its functions, via regulation of Rap1 activity, include regulation of cell adhesion, suppression of cell proliferation and metastasis. In other epithelial cancers, loss of Rap1Gap has been linked to epithelial to mesenchymal transition (EMT) and invasion. To begin study of Rap1Gap in breast cancer progression, we performed immunohistochemistry. We show strong Rap1Gap staining in DCIS with little to no expression in myoepithelial and stromal components. Immunoblotting results show that Rap1Gap levels in MCF10.CA1d cells (a model of invasive carcinoma) are reduced compared to those in MCF10.DCIS cells. Rap1Gap expression is low in additional basal breast cancer cell lines, but high in luminal ER+ /PR+ cell lines. We propose that over-expression of Rap1Gap may serve as a tumor suppressive mechanism in DCIS; subsequent reduction of Rap1Gap may be a switch for EMT and progression to an invasive phenotype. Citation Format: Seema Shah, Kingsley Osuala, Shihong Mao, Quanwen Li, Bonnie Sloane, Stephen Krawetz, Raymond R. Mattingly. Exploring the role of Rap1Gap in the progression from DCIS to invasive breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2014-3150

Abstract 3586: HIF-1α from tumor-surround fibroblasts contributes to breast cancer malignant progression

Abstract As tumor-surrounding stromal cells (such as fibroblasts) are integrate components of the tumor environment that together as an entity plays critical roles in the tumor progression, we investigated whether HIF-1α from tumor-surrounding fibroblasts plays a critical role in breast cancer (BCa) malignant progression. We co-cultured BCa 4T1/GFP cells with MEF fibroblasts that either expressed or lacked the HIF-1α gene in a 3D culture system established in our lab. The mixed cells (BCa:MEF) grew in a suspension manner and formed aggregates/spheroids. We found that HIF-1α plays a role under hypoxia in the 3D BCa cells' protrusion formation, an established marker for invasive, aggressively malignant tumor cells that start to invade the surrounding matrix. These protrusions (fluorescent) only appeared in mixed-cell aggregates containing HIF-1α wild-type MEF fibroblasts; but not appear in mixed-cell aggregates containing HIF-1α knockout MEF fibroblasts, implying HIF-1α from fibroblasts (MEF) plays a critical role in inducing surrounding epithelial BCa cells to form protrusions, an indicator that tumor cells have become invasive. Moreover, we performed transwell overnight invasion assay of BCa LM2 cells with and without cocultured MEF under both normoxia (21% O2) and hypoxia (1% O2). We found that MEF significantly promoted BCa invade through matrigel, and HIF-1α WT MEF promoted BCa invasion more than HIF-KO MEF did under hypoxia. Furthermore, by using Boyden's chamber-based cell migration assay, we found that migration of HIF-1α WT BCa (MMTC-PyT) cells increased more than two fold under hypoxia than normoxia. However, this hypoxia-induced increase in migration disappeared if HIF-1α KO counterparts were used. The loss of this hypoxia-induced migration in HIF-1α KO cells can be rescued by exogenous HIF-1α with Ad-HIF-1α transduction. A similar pattern of HIF-1α-dependent, hypoxia-stimulated cell migration was observed in fibroblasts when we analyzed HIF-1α WT and KO MEF cells, demonstrating that hypoxia stimulated a HIF-1α-dependent cell migration in both BCa and MEF cells. Together, these results indicate that HIF-1α from tumor-surrounding fibroblasts plays critical roles in promoting BCa malignant progression. Citation Format: Yi Lu, Jun Zhang. HIF-1α from tumor-surround fibroblasts contributes to breast cancer malignant progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2014-3586

Abstract 173: Tumor proximal mesenchymal stem cells initiate a pro-metastatic microRNA regulatory network which acts via convergent targeting of the speech-associated transcriptional repressor FOXP2

Abstract Tumor progression to metastasis is largely responsible for breast cancer mortalities, but the mechanisms underlying this process remain among the least understood in oncology. Cell-intrinsic mechanisms, such as accumulating genetic lesions, are well appreciated drivers of malignant progression; however, substantial evidence now supports a similarly critical role for cell-extrinsic stimuli, driving pro-malignant gene expression changes through epigenetic mechanisms. Such external stimuli derive from interactions with the tumor-associated stroma, a distinctive milieu of non-transformed stromal cells recruited to the tumor site by the growing neoplasm. Proximal interactions between cancer cells and components of this tumor-associated stroma have been demonstrated to drive angiogenesis, invasion and metastasis. Understanding these interactions will inform our knowledge of tumor progression and metastasis in general, and presents potential for developing therapeutic approaches based on interdicting the tumor-stroma crosstalk. Mesenchymal stem cells (MSCs) are ambulatory stromal progenitor cells that are normally dispatched to wound sites to aid in the repair of damaged tissues. MSCs similarly home with great affinity to tissues perturbed growing epithelial neoplasms, where they have been found to serve as stromal catalysts of carcinoma progression and metastasis in multiple experimental tumor models of a variety of human cancer contexts including lung, breast, melanoma, prostate, and colon. Despite expanding knowledge of how tumor-associated MSCs contribute to tumor malignancy, the programs and pathways deregulated in cancer cells as a consequence of their interactions with MSCs remain incompletely understood. We conducted genome-wide microRNA (miRNA) profiling analyses on MSC-exposed breast cancer cells (BCCs) to investigate how miRNA deregulation might contribute to malignant breast cancer progression. Our investigation revealed that MSCs cause aberrant expression of a distinct set of miRNAs that accelerate metastasis and are sufficient to provide cancer cells with enhanced tumor initiation capacities, including phenotypes and markers characteristic of putative cancer stem cells. We demonstrate that these miRNAs collectively constitute a regulatory network whose members converge upon a common target: the speech-associated transcriptional repressor FOXP2. FOXP2 knockdown phenocopied increases in metastasis and tumor-initiating ability of BCCs, including CSC-consistent markers, genes and phenotypes. Importantly, elevated MSC-induced miRNA levels and depressed FOXP2 levels were observed in human breast cancers as correlating with increased malignancy. Altogether, our results implicate FOXP2 and its MSC-induced miRNA regulators as novel determinants of malignant breast cancer progression. Citation Format: Benjamin Cuiffo, Antoine Campagne, George W. Bell, Evan Lien, Manoj K. Bhasin, Odette Mariani, Anne Vincent-Salomon, Antoine Karnoub. Tumor proximal mesenchymal stem cells initiate a pro-metastatic microRNA regulatory network which acts via convergent targeting of the speech-associated transcriptional repressor FOXP2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2014-173

441. Suppression of Hypoxia/HIF-1° Promoted Breast Cancer Malignant Progression by p16/ INK4a

441. Suppression of Hypoxia/HIF-1° Promoted Breast Cancer Malignant Progression by p16/ INK4a

Role of carbohydrate response element-binding protein (ChREBP) in generating an aerobic metabolic phenotype and in breast cancer progression.

Background:The lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) may play a key role in malignant progression of breast cancer by allowing metabolic adaptations to take place in response to changes in oxygenation.Methods:Immunohistochemical analysis of ChREBP was carried out in human breast tumour tissue microarrays representative of malignant progression from normal breast through to metastatic cancer. The ChREBP protein and mRNA expressions were then analysed in a series of breast cancers for correlative analysis with common and breast-specific hypoxia signatures, and survival.Results:In invasive ductal carcinoma, ChREBP correlated significantly with mean ‘downregulated' hypoxia scores (r=0.3, P<0.015, n=67) and in two distinct breast progression arrays, ChREBP protein also increased with malignant progression (P<0.001). However, bioinformatic analysis of a large data set (2136 cases) revealed an apparent reversal in the relationship between ChREBP mRNA level and clinical outcome – not only being significantly correlated with increased survival (log rank P<0.001), but also downregulated in malignant tissue compared with adjacent normal tissue.Conclusion:The ChREBP expression may be reflective of an aerobic metabolic phenotype that may conflict with hypoxia-induced signalling but provide a mechanism for growth at the oxygenated edge of the tumours.

Analysis of HOX Gene Expression Patterns in Human Breast Cancer

HOX genes are highly conserved transcription factors that determine the identity of cells and tissues along the anterior-posterior body axis in developing embryos. Aberrations in HOX gene expression have been shown in various tumors. However, the correlation of HOX gene expression patterns with tumorigenesis and cancer progression has not been fully characterized. Here, to analyze putative candidate HOX genes involved in breast cancer tumorigenesis and progression, the expression patterns of 39 HOX genes were analyzed using breast cancer cell lines and patient-derived breast tissues. In vitro analysis revealed that HOXA and HOXB gene expression occurred in a subtype-specific manner in breast cancer cell lines, whereas most HOXC genes were strongly expressed in most cell lines. Among the 39 HOX genes analyzed, 25 were chosen for further analysis in malignant and non-malignant tissues. Fourteen genes, encoding HOXA6, A13, B2, B4, B5, B6, B7, B8, B9, C5, C9, C13, D1, and D8, out of 25 showed statistically significant differential expression patterns between non-malignant and malignant breast tissues and are putative candidates associated with the development and malignant progression of breast cancer. Our data provide a valuable resource for furthering our understanding of HOX gene expression in breast cancer and the possible involvement of HOX genes in tumor progression.

A novel 2-pyrone derivative, BHP, impedes oncogenic KRAS-driven malignant progression in breast cancer

Elevated KRAS expression has been frequently associated with cancer progression including breast cancer; however, therapeutic approaches targeting KRAS have been widely unsuccessful and KRAS mutant cancers remain unsolved problem in cancer therapy. In this study, we found that a new 2-pyrone derivative, 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one (BHP) can block KRAS-driven breast cancer progression. Importantly, treatment with BHP effectively suppressed the migratory and invasive properties along with epithelial–mesenchymal transition (EMT) in MDA-MB231 breast cancer cells that carry oncogenic KRAS and mesenchymal malignant phenotypes. In parallel, BHP also sensitized the cells to anticancer treatment. Consistently, forced-expression of oncogenic KRAS bestowed the migratory and invasive properties, mesenchymal transition and resistance to anticancer treatment into normal human mammalian breast cells MCF10A and relatively non-malignant MCF7 and SK-BR3 breast cancer cells; however, treatment with BHP blocked those KRAS-induced malignant phenotypes. Notably, BHP interfered the interaction of KRAS with Raf-1 in concentration-dependent manner, thereby blocking the downstream effectors of KRAS signaling that is PI3K/AKT and ERK. Taken together, our findings indicate that the BHP, an α-pyrone derivative, suppresses malignant breast cancer progression by targeting of oncogenic KRAS signaling pathways.

136. Inhibition of Breast Cancer Malignant Progression by Neutralizing Hypoxia-Induced Cell Motility

136. Inhibition of Breast Cancer Malignant Progression by Neutralizing Hypoxia-Induced Cell Motility

Sphingosine‐1‐phosphate (S1P) Signaling for Breast Cell Invasion

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1‐phosphate (S1P), acting on the G‐protein‐coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase‐9 (MMP‐9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP‐9 by S1P. In addition, activation of extracellular‐signal‐regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine‐protein kinase (Akt) are involved in the process of S1P‐mediated induction of MMP‐9 expression and invasion. Activation of the S1P receptor S1P3 and Gαq are required for S1P‐induced invasive/migratory responses, suggesting that the enhancement of S1P‐mediated invasiveness is triggered by the specific coupling of S1P3 to the eterotrimeric Gαq subunit. Activation of phospholipase C‐β4 and intracellular Ca2+ release are required for S1P‐induced MMP‐9 upregulation. Taken together, this study demonstrated that S1P regulates MMP‐9 induction and invasiveness through coupling of S1P3 and Gαq in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.

The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

The polypeptide hormone prolactin (PRL) stimulates breast epithelial cell growth, differentiation, and motility through its cognate receptor, PRLr. PRLr is expressed in most breast cancers; however, its exact role remains elusive. Our laboratory previously described a novel mode of PRLr signaling in which Stat5a-mediated transcription is regulated through ligand-induced phosphorylation of the PRLr transactivation domain (TAD). Herein, we used a PRLr transactivation-deficient mutant (PRLrYDmut) to identify novel TAD-specific target genes. Microarray analysis identified 120 PRL-induced genes up-regulated by wild type but not PRLrYDmut. Compared with control, PRLr expression significantly induced expression of approximately 4700 PRL-induced genes, whereas PRLrYDmut ablated induction of all but 19 of these genes. Ingenuity pathway analysis found that the PRLr TAD most profoundly affected networks involving cancer and proliferation. In support of this, PRLrYDmut expression reduced anchorage-dependent and anchorage-independent growth. In addition, pathway analysis identified a link between the PRLr TAD and the estrogen and progesterone receptors (ERα/PR). Although neither ERα nor PR was identified as a PRL target gene, a TAD mutation significantly impaired ERα/PR expression and estrogen responsiveness. TMA analysis revealed a marked increase in nuclear, but not cytoplasmic, PRLr TAD phosphorylation as a function of neoplastic progression. We propose that PRLr TAD phosphorylation contributes to breast cancer pathogenesis, in part through regulation of ERα and PR, and has potential utility as a biomarker in this disease.

Expression and significance of phosphorylated Girdin in breast cancer

To detect the expression of phosphorylated girdin (p-girdin) in breast cancer and its association with pathological characteristics and molecular subtypes of breast cancer. Immunohistochemical SP staining was used to investigate the expression of p-girdin in 27 cases of lobular hyperplasia of mammary gland, 61 cases of ductal carcinoma in situ (DCIS), and 94 cases of non-special type invasive carcinoma (IDC-NOS) of breast. p-girdin was located in the cell cytoplasm and (or) nuclei in breast cancer. There was statistically a very significant difference among lobular hyperplasia, DCIS and IDC-NOS (χ2=26.724, P<0.001). Its cytoplasmic and nuclear reactivity were 25.9% (7/27), 39.3% (24/61), and 66.0% (62/94), respectively. The expression of p-girdin was positively associated with pathologic stage (r=0.204, P=0.049), lymph node metastasis (r=0.212, P=0.041) and HER2/neu (r=0.248, P=0.016). But no significant association was identified between p-girdin expression and histological grade (r=-0.015, P=0.918), age of patients (r=-0.011, P=0.918), tumor size (r=0.075, P=0.471), ER(r=0.071, P=0.498), PR (r=-0.050, P=0.634). Mann-Whitney test showed that p-girdin expression in luminal A, luminal B, triple negative and HER-2(+) type was significantly different (χ2=14.017, P=0.003). Among the four types, its positive rate was 55.8% (24/43), 95.8% (23/24), 66.7% (10/15), and 41.7% (5/12), respectively. p-Girdin expression is closely correlated with the malignant progression of breast cancer. Its expression may have clinical value as a new target for the treatment of breast cancer.