Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and −2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Gαi/o and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.
thrombin; GPCR; metalloprotease; MDA-MB-231; G protein
The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3ζ overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3ζ overexpression-mediated Akt phosphorylation and the potential impact on breast cancer progression. We found that 14-3-3ζ overexpression was significantly (P = 0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3ζ overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3ζ expression by siRNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3ζ enhanced Akt phosphorylation through activation of PI3K. Mechanistically, 14-3-3ζ bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3 binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3ζ-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3ζ binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage independent growth and enhanced stress induced apoptosis. These findings revealed a novel mechanism by which 14-3-3ζ overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.
14-3-3ζ; breast cancer; PI3K; Akt
Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling.
We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics.
MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2. Furthermore, we could demonstrate that suppression of AhR functions through anti-AhR siRNA or MNF causes the recovery of ERalpha functions.
One of the main causes for AhR overexpression in these MCF breast cancer cells appears to be the loss of ERalpha functions. This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR.
Vascular endothelial growth factor receptor-3 is a receptor tyrosine kinase that is overexpressed in some human carcinomas, but its role in tumorigenesis has not been fully elucidated. We examined VEGFR-3 expression in normal, nonneoplastic and early stage malignant breast tissues and have shown that VEGFR-3 upregulation in breast cancer preceded tumor cell invasion, suggesting that VEGFR-3 may function as a survival signal. We characterized the biological effects of VEGFR-3 over-expression in human breast cancer cells based on two approaches: gain of function by overexpressing VEGFR-3 in MCF-7 breast cancer cells and loss of function by RNAi-mediated silencing of VEGFR-3 in MCF-7-VEGFR-3 and BT474 cells.
VEGFR-3 overexpression increased cellular proliferation by 40% when MCF7-VEGFR-3 cells were compared to parental MCF7 cells, and proliferation was reduced by more than 40% when endogenous VEGFR-3 was downregulated in BT474 cells. VEGFR-3 overexpression promoted a three-fold increase in motility and invasion and both motility and invasion were inhibited by downregulation of VEGFR-3. Furthermore, VEGFR-3 overexpression promoted cellular survival under stress conditions induced by staurosporine treatment and led to anchorage-independent growth.
VEGFR-3 overexpression dramatically increased tumor formation in both hormone-dependent and independent xenograft models. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger than control xenografts. Finally, downregulation of VEGFR-3 expression in both xenograft model cell lines led to a significant reduction of tumor growth. For the first time, we have demonstrated that VEGFR-3 overexpression promotes breast cancer cell proliferation, motility, survival, anchorage-independent growth and tumorogenicity in the absence of ligand expression.
vascular endothelial growth factor receptor-3; VEGFR-3; Flt-4; receptor tyrosine kinases; breast cancer; tumorigenicity; carcinogenesis
Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-β) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-β1 on breast cancer cells in the presence or absence of overexpressed HER-2.
Cell proliferation assays were used to determine the effect of TGF-β on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-β1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-β signaling pathway was assessed using TGF-β1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays.
We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-β1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-β in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-β induced pro-invasive and pro-metastatic gene signature.
HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-β. In contrast, HER-2 and TGF-β signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.
Biomarkers, commonly expressed in breast cancer cells, may be correlated with their expression in breast skin of the same subjects.
The expression of biomarkers in specimens from 33 breast tumours and breast skin from the same subject and from 32 normal controls was studied using immunohistochemical techniques.
(1) In normal women, there are significant correlations between the levels of expression of cyclin D1, bcl‐2 and p53 in normal breast epithelial cells and breast skin epithelial cells. (2) These patterns of biomarker expression in normal women are similar in breast cancer and breast skin epithelial cells of women with invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS), but are at significantly higher levels in both breast cancer cells and skin from the same subjects. (3) In normal women, human epidermal growth factor receptor 2 (HER‐2) is not expressed in either breast epithelial cells or skin epithelial cells. (4) HER‐2 is expressed in the breast skin of some subjects with HER‐2‐positive breast cancer. (5) Positive oestrogen receptor alpha expression occurs significantly more frequently in the breast skin of women with IDC and DCIS than in normal controls.
The influence of localised breast cancer seems to be systemic, and leads to changes in skin and hair.
Molecular markers for predicting breast cancer patients at high risk of recurrence are urgently needed for more effective disease management. The impact of alterations in extracellular matrix components on tumor aggressiveness is under intense investigation. Overexpression of Transglutaminase 2 (TG2), a multifunctional enzyme, in cancer cells impacts epithelial mesenchymal transition, growth, invasion and interactions with tumor microenvironment. The objective of our study is to determine the clinical relevance of stromal TG2 overexpression and explore its potential to identify breast cancers at high risk of recurrence.
This retrospective study is based on immunohistochemical analysis of TG2 expression in normal breast tissues (n = 40) and breast cancers (n = 253) with clinical, pathological and follow-up data available for up to 12 years. TG2 expression was correlated with clinical and pathological parameters as well as disease free survival (DFS) of breast cancer patients.
Stromal TG2 overexpression was observed in 114/253 (45.0%) breast cancer tissues as compared to breast normal tissues. Among invasive ductal carcinomas (IDC) of the breast, 97/168 (57.7%) showed strong TG2 expression in tumor stroma. Importantly, IDC patients showing stromal TG2 accumulation had significantly reduced DFS (mean DFS = 110 months) in comparison with patients showing low expression (mean DFS = 130 months) in Kaplan-Meier survival analysis (p<0.001). In Cox multivariate regression analysis, stromal TG2 accumulation was an independent risk factor for recurrence (p = 0.006, Hazard’s ratio, H.R. = 3.79). Notably, these breast cancer patients also showed immunostaining of N-epsilon gamma-glutamyl lysine amino residues in tumor stroma demonstrating the transamidating activity of TG2.
Accumulation of TG2 in tumor stroma is an independent risk factor for identifying breast cancer patients at high risk of recurrence. TG2 overexpression in tumor stroma may serve as a predictor of poor prognosis for IDC of the breast.
ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers including breast carcinomas. NGAL can inhibit erythroid cell production leading to anemia. Anemia usually occurs in cancer patients and negatively impacts quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we demonstrate that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor kappa B (NF-κB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by an NF-κB inhibitor and an ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-κB activation is involved in cancer-related anemia, and ErbB2, NF-κB, NGAL pathway may serve as potential therapeutic targets for cancer-related anemia.
ErbB2; neutrophil gelatinase-associated lipocalin (NGAL); nuclear factor kappa B (NF-κB); anemia; breast cancer
Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells.
Quantitative real time RT-PCR analysis using primers specific for t-DARPP demonstrated overexpression of t-DARPP in 36% of breast cancers (13/36) as opposed to absent to very low t-DARPP expression in normal breast tissue (p < 0.05). The mRNA overexpression of t-DARPP was overwhelmingly observed in ductal carcinomas, including invasive ductal carcinomas and intraductal carcinomas, rather than other types of breast cancers. The immunohistochemistry analysis of DARPP-32/t-DARPP protein(s) expression in breast cancer tissue microarray that contained 59 tumors and matched normal tissues when available indicated overexpression in 35.5% of primary breast tumors that were more frequent in invasive ductal carcinomas (43.7%; 21/48). In vitro studies showed that stable overexpression of t-DARPP in MCF-7 cells positively regulated proliferation and anchorage-dependent and -independent growth. Furthermore, this effect was concomitant with induction of phosphorylation of AKTser473 and its downstream target phosphoser9 GSK3β, and increased Cyclin D1 and C-Myc protein levels. The knockdown of endogenous t-DARPP in HCC1569 cells led to a marked decrease in phosphorylation of AKTsser473 and GSK3βser9. The use of PI3K inhibitor LY294002 or Akt siRNA abrogated the t-DARPP-mediated phosphorylation of AKTser473 and led to a significant reduction in cell growth.
Our findings underscore the potential role of t-DARPP in regulating cell growth and proliferation through PI3 kinase-dependent mechanism.
Aberrant methylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. However, its impact on tumor development is not fully determined, and its potential as a diagnostic biomarker remains to be validated. Methylation profiling of invasive breast carcinoma has been largely explored. Conversely, very little and sparse information is available on early-stage breast cancer. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, we have analyzed the DNA methylation profile of ductal carcinoma in situ, a premalignant breast lesion with a great potential to progress toward invasive carcinoma.
We have utilized a comprehensive and sensitive array-based DNA mapping technique, the methylated-CpG island recovery assay, to profile the DNA methylation pattern in ductal carcinoma in situ. Differential methylation of CpG islands was compared genome-wide in tumor DNA versus normal DNA utilizing a statistical linear model in the LIMMA software package.
Using this approach, we have identified 108 significant CpG islands that undergo aberrant DNA methylation in ductal carcinoma in situ and stage I breast tumors, with methylation frequencies greater than or comparable with those of more advanced invasive carcinoma (50% to 93%). A substantial fraction of these hypermethylated CpG islands (32% of the annotated CpG islands) is associated with several homeobox genes, such as the TLX1, HOXB13, and HNF1B genes. Fifty-three percent of the genes hypermethylated in early-stage breast cancer overlap with known Polycomb targets and include homeobox genes and other developmental transcription factors.
We have identified a series of new potential methylation biomarkers that may help elucidate the underlying mechanisms of breast tumorigenesis. More specifically, our results are suggestive of a critical role of homeobox gene methylation in the insurgence and/or progression of breast cancer.
Elafin is an elastase-specific inhibitor with increased transcription in normal mammary epithelial cells compared to mammary carcinoma cells. In this report, we test the hypothesis that inhibition of elastase, through induction of elafin, leads to inhibition of human breast cancer cell viability and, therefore, predicts survival in breast cancer patients.
Panels of normal and immortalized breast epithelial cells, along with breast carcinoma cells, were used to examine the impact of adenoviral-mediated elafin expression or shRNA-mediated inhibition of elastase on the growth of cells and xenografts in nude mice. To determine the prognostic significance of decreased elafin in patients with invasive breast cancer, previously published gene array datasets were interrogated.
Elafin expression had no effect on non-tumorigenic cells but resulted in marked inhibition of cell growth in breast cancer cell lines. Control-treated xenografts generated a tumor burden that necessitated sacrifice within one month of initial treatment, whereas xenograft-bearing mice treated with Ad-Elafin were alive at eight months with marked reduction in tumor growth. Elastase inhibition mimicked these results, showing decreased tumor cell growth in vitro and in vivo. Low expression of elafin gene correlated with significantly reduced time to relapse, and when combined with high expression of elastase gene was associated with decreased survival in breast cancer patients.
Our data suggest that elafin plays a direct role in the suppression of tumors through inhibition of elastase and thus serves as a prognostic indicator for breast cancer patients.
Protein Kinase D1 is downregulated in its expression in invasive ductal carcinoma of the breast and in invasive breast cancer cells, but its functions in normal breast epithelial cells is largely unknown. The epithelial phenotype is maintained by cell-cell junctions formed by E-cadherin. In cancer cells loss of E-cadherin expression contributes to an invasive phenotype. This can be mediated by SNAI1, a transcriptional repressor for E-cadherin that contributes to epithelial-to-mesenchymal transition (EMT).
Here we show that PKD1 in normal murine mammary gland (NMuMG) epithelial cells is constitutively-active in its basal state and prevents a transition to a mesenchymal phenotype. Investigation of the involved mechanism suggested that PKD1 regulates the expression of E-cadherin at the promoter level through direct phosphorylation of the transcriptional repressor SNAI1. PKD1-mediated phosphorylation of SNAI1 occurs in the nucleus and generates a nuclear, inactive DNA/SNAI1 complex that shows decreased interaction with its co-repressor Ajuba. Analysis of human tissue samples with a newly-generated phosphospecific antibody for PKD1-phosphorylated SNAI1 showed that regulation of SNAI1 through PKD1 occurs in vivo in normal breast ductal tissue and is decreased or lost in invasive ductal carcinoma.
Our data describe a mechanism of how PKD1 maintains the breast epithelial phenotype. Moreover, they suggest, that the analysis of breast tissue for PKD-mediated phosphorylation of SNAI1 using our novel phosphoS11-SNAI1-specific antibody may allow predicting the invasive potential of breast cancer cells.
Inflammatory breast cancer (IBC) is a distinct and aggressive form of locally advanced breast cancer. IBC is highly angiogenic, invasive, and metastatic at its inception. Previously, we identified specific genetic alterations of IBC that contribute to this highly invasive phenotype. RhoC GTPase was overexpressed in 90% of archival IBC tumor samples, but not in stage-matched, non-IBC tumors. To study the role of RhoC GTPase in contributing to an IBC-like phenotype, we generated stable transfectants of human mammary epithelial cells overexpressing the RhoC gene, and studied the effect of RhoC GTPase overexpression on the modulation of angiogenesis in IBC. Levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) were significantly higher in the conditioned media of the HME-RhoC transfectants than in the untransfected HME and HME-β-galactosidase control media, similar to the SUM149 IBC cell line. Inhibition of RhoC function by introduction of C3 exotransferase decreased production of angiogenic factors by the HME-RhoC transfectants and the SUM149 IBC cell line, but did not affect the control cells. These data support the conclusion that overexpression of RhoC GTPase is specifically and directly implicated in the control of the production of angiogenic factors by IBC cells.
inflammatory breast cancer; human mammary epithelial cells; RhoC GTPase; angiogenesis; angiogenic factors
The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.
To evaluate the expression of cathepsin K (CTSK) and CXCL14 in stromal and epithelial cells in human breast tumor progression.
We did immunohistochemical analyses of CTSK and CXCL14 expression in normal breast tissue, biopsy sites, benign lesions, ductal carcinoma in situ, and invasive breast tumors of different stages. Expression patterns were related to histopathologic characteristics of the tumors and clinical outcome. The effect of CTSK+ breast stromal fibroblasts on CTSK-breast cancer cells was assessed in coculture.
Epithelial expression of CTSK was rarely detectedin any of the tissue samples analyzed, whereas CXCL14-positive epithelial cells were found in all tissue types. The expression of CXCL14 was not associated with any tumor or patient characteristics analyzed. Stromal CTSK expression was significantly higher in invasive compared with in situ carcinomas, and in one of the two data sets analyzed, it correlated with higher tumor stage. Among all samples examined, the highest stromal CTSK levels were detected in biopsy sites. Neither epithelial nor stromal expression of CTSK was significantly associated with recurrence-free or overall survival. Coculture of CTSK+ fibroblastsenhanced the invasion of CTSK-breast tumor epithelial cells and this was blocked by CTSK inhibitors.
CTSK may function as a paracrine factor in breast tumorigenesis. CTSK+ fibroblasts may play a role in tumor progression by promoting the invasiveness of tumor epithelial cells. The possibility that CTSK inhibitors may have a clinical role in decreasing the risk of tumor progression merits further investigation.
A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as 3- dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional up-regulation of multiple components of the Notch survival pathway. Importantly, luminal filling required up-regulation of a signaling pathway comprising Notch3, its cleaved intracellular domain (NICD) and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and Cyclin D1. In line with HER2- Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report up-regulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.
breast cancer; DCIS; growth factor; spheroids; receptor tyrosine kinase; signal transduction
Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma.
We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein.
Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use.
Inflammatory cytokines within the tumor microenvironment are linked to progression in breast cancer. Interleukin- (IL-) 19, part of the IL-10 family, contributes to a range of diseases and disorders, such as asthma, endotoxic shock, uremia, psoriasis, and rheumatoid arthritis. IL-19 is expressed in several types of tumor cells, especially in squamous cell carcinoma of the skin, tongue, esophagus, and lung and invasive duct carcinoma of the breast. In breast cancer, IL-19 expression is correlated with increased mitotic figures, advanced tumor stage, higher metastasis, and poor survival. The mechanisms of IL-19 in breast cancer have recently been explored both in vitro and in vivo. IL-19 has an autocrine effect in breast cancer cells. It directly promotes proliferation and migration and indirectly provides a microenvironment for tumor progression, which suggests that IL-19 is a prognostic marker in breast cancer and that antagonizing IL-19 may have therapeutic potential.
The TGF-β signaling pathway has a complex role in regulating mammary carcinogenesis. Here we demonstrate that the type III TGF-β receptor (TβRIII, or betaglycan), a ubiquitously expressed TGF-β coreceptor, regulated breast cancer progression and metastasis. Most human breast cancers lost TβRIII expression, with loss of heterozygosity of the TGFBR3 gene locus correlating with decreased TβRIII expression. TβRIII expression decreased during breast cancer progression, and low TβRIII levels predicted decreased recurrence-free survival in breast cancer patients. Restoring TβRIII expression in breast cancer cells dramatically inhibited tumor invasiveness in vitro and tumor invasion, angiogenesis, and metastasis in vivo. TβRIII appeared to inhibit tumor invasion by undergoing ectodomain shedding and producing soluble TβRIII, which binds and sequesters TGF-β to decrease TGF-β signaling and reduce breast cancer cell invasion and tumor-induced angiogenesis. Our results indicate that loss of TβRIII through allelic imbalance is a frequent genetic event during human breast cancer development that increases metastatic potential.
Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11–12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.
Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.
TWIST; ERα; NuRD complex; gene repression; breast cancer
cDNA microarray-derived expression profiles of MMTV-Wnt-1 and MMTV-Neu transgenic mice reveal several hundred genes to be differentially expressed at each stage of breast tumor development.
In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development.
We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes.
We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression.
The protein kinase, PAK1, is overexpressed in human breast cancer and may contribute to malignancy through induction of proliferation and invasiveness. In this study, we examined the role of PAK1 in the survival of detached MCF10A breast epithelial cells to test whether it may also regulate the early stages of neoplasia. MCF10A cells undergo anoikis, as measured by the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP), after more than 8 hours of detachment. Endogenous Akt, PAK1, and BAD are phosphorylated in attached MCF10A cells, but these phosphorylation events are all lost during the first 8 hours of detachment. Expression of constitutively active PAK1 or Akt suppresses the cleavage of caspase 3 and PARP in detached MCF10A cells. Co-overexpression of active PAK1 with dominant-negative Akt, or of active Akt with dominant-negative PAK1, still suppresses anoikis. Thus, Akt and PAK1 enhance survival through pathways that are at least partially independent. PAK1-dependent regulation of anoikis is likely to occur early in the apoptotic cascade as expression of dominant-negative PAK1 increased the cleavage of the upstream caspase 9, while constitutively active PAK1 inhibited caspase 9 activation. These results support a role for activated PAK1 in the suppression of anoikis in MCF10A epithelial cells.
Breast carcinoma; apoptosis; cell survival; caspase activation; protein kinases
Ductal carcinoma in situ (DCIS) is characterized by ductal epithelial cells that have filled the luminal space of the breast duct and survive despite loss of extracellular matrix contact. In normal epithelial cells, the loss of such contact triggers a form of apoptosis known as detachment-induced apoptosis or “anoikis.” TMS1/ASC is a bipartite adaptor molecule that participates in inflammatory and apoptotic signaling pathways. Epigenetic silencing of TMS1 has been observed in a significant proportion of human breast and other cancers, but the mechanism by which TMS1 silencing contributes to carcinogenesis is unknown. Here we examined the role of TMS1 in anoikis. We found that TMS1 expression is induced in response to loss of substratum interactions in breast epithelial cells. siRNA-mediated knockdown of TMS1 leads to anoikis resistance, due in part to the persistent activation of ERK and an impaired ability to upregulate the BH3-only protein Bim. We further show that the detachment-induced cleavage of procaspase-8, a newly described mediator of cellular adhesion, is significantly inhibited in the absence of TMS1. These data demonstrate a novel upstream role for TMS1 in the promotion of anoikis, and suggest that silencing of TMS1 may contribute to the pathogenesis of breast cancer by allowing epithelial cells to bypass cell death in the early stages of breast cancer development. This conclusion is supported by in vivo data showing that TMS1 is selectively downregulated in the aberrant epithelial cells filling the lumen of the breast duct in a subset of primary DCIS lesions.
DCIS; breast cancer; apoptosis; caspase-8; Bim
The molecular events that lead to malignant transformation and subsequent metastasis of breast carcinoma include alterations in the cells at genome, transcriptome and proteome levels. In this study, we used publicly available gene expression databases to identify those candidate genes which are upregulated at the mRNA level in breast cancers but have not been systematically validated at the protein level. Based on an extensive literature search, we identified ribosome binding protein 1 (RRBP1) as a candidate that is upregulated at the mRNA level in five different studies but its protein expression had not been investigated. Immunohistochemical labeling of breast cancer tissue microarrays was carried out to determine the expression of RRBP1 in a large panel of breast cancers. We found that RRBP1 was overexpressed in 84% (177/219) of breast carcinoma cases tested. The subcellular localization of RRBP1 was mainly observed to be in the cytoplasm with intense staining in the perinuclear region. Our findings suggest that RRBP1 is an interesting molecule that can be further studied for its potential to serve as a breast cancer biomarker. This study also demonstrates how the integration of biological data from available resources in conjunction with systematic evaluation approaches can be successfully applied to clinical proteomics.
Breast neoplasms; ES130; p180; 180 kDa ribosome receptor homolog; Endoplasmic reticulum membrane protein; Immunohistochemistry; Biomarker; Early detection