Crk adaptor proteins act as key signaling integrators for breast tumorigenesis

Introduction

CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo.

Methods

The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer.

Results

A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation.

Conclusions

These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.     

Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

Introduction

Human models of noninvasive breast tumors are limited, and the existing in vivo models do not mimic inter- and intratumoral heterogeneity. Ductal carcinoma in situ (DCIS) is the most common type (80%) of noninvasive breast lesions. The aim of this study was to develop an in vivo model whereby the natural progression of human DCIS might be reproduced and studied. To accomplish this goal, the intraductal human-in-mouse (HIM) transplantation model was developed. The resulting models, which mimicked some of the diversity of human noninvasive breast cancers in vivo, were used to show whether subtypes of human DCIS might contain distinct subpopulations of tumor-initiating cells.

Methods

The intraductal models were established by injection of human DCIS cell lines (MCF10DCIS.COM and SUM-225), as well as cells derived from a primary human DCIS (FSK-H7), directly into the primary mouse mammary ducts via cleaved nipple. Six to eight weeks after injections, whole-mount, hematoxylin and eosin, and immunofluorescence staining were performed to evaluate the type and extent of growth of the DCIS-like lesions. To identify tumor-initiating cells, putative human breast stem/progenitor subpopulations were sorted from MCF10DCIS.COM and SUM-225 with flow cytometry, and their in vivo growth fractions were compared with the Fisher's Exact test.

Results

Human DCIS cells initially grew within the mammary ducts, followed by progression to invasion in some cases into the stroma. The lesions were histologically almost identical to those of clinical human DCIS. This method was successful for growing DCIS cell lines (MCF10DCIS.COM and SUM-225) as well as a primary human DCIS (FSK-H7). MCF10DCIS.COM represented a basal-like DCIS model, whereas SUM-225 and FSK-H7 cells were models for HER-2⁺ DCIS. With this approach, we showed that various subtypes of human DCIS appeared to contain distinct subpopulations of tumor-initiating cells.

Conclusions

The intraductal HIM transplantation model provides an invaluable tool that mimics human breast heterogeneity at the noninvasive stages and allows the study of the distinct molecular and cellular mechanisms of breast cancer progression.     

Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

Introduction

Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs.

Methods

We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24^low/CD44⁺ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers.

Results

High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24^low/CD44⁺ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-β) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers.

Conclusions

Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-β and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.     

Focal adhesion kinase contributes to proliferative potential of ErbB2 mammary tumour cells but is dispensable for ErbB2 mammary tumour induction in vivo

Introduction

Neu (HER2/ErbB2) is overexpressed in 25% to 30% of human breast cancer, correlating with a poor prognosis. Researchers in previous studies who used the mouse mammary tumor virus Neu-transgenic mouse model (MMTV-Neu) demonstrated that the Neu-YB line had increased production of CXCL12 and increased metastasis, whereas the Neu-YD line had decreased metastasis. In this study, we examined the role of increased production of CXCL12 in tumor cell invasion and malignancy.

Methods

We studied invasion in the tumor microenvironment using multiphoton intravital imaging, in vivo invasion and intravasation assays. CXCL12 signaling was altered by using the CXCR4 inhibitor AMD3100 or by increasing CXCL12 expression. The role of macrophage signaling in vivo was determined using a colony-stimulating factor 1 receptor (CSF-1R) blocking antibody.

Results

The Neu-YD strain was reduced in invasion, intravasation and metastasis compared to the Neu-YB and Neu deletion mutant (activated receptor) strains. Remarkably, in the Neu-YB strain, in vivo invasion to epidermal growth factor was dependent on both CXCL12-CXCR4 and CSF1-CSF-1R signaling. Neu-YB tumors had increased macrophage and microvessel density. Overexpression of CXCL12 in rat mammary adenocarcinoma cells increased in vivo invasion as well as microvessel and macrophage density.

Conclusions

Expression of CXCL12 by tumor cells results in increased macrophage and microvessel density and in vivo invasiveness.     

Silencing of GM3 synthase suppresses lung metastasis of murine breast cancer cells

Background

Gangliosides are sialic acid containing glycosphingolipids that are ubiquitously distributed on vertebrate plasma membranes. GM3, a precursor for most of the more complex ganglioside species, is synthesized by GM3 synthase. Although total ganglioside levels are significantly higher in breast tumor tissue than in normal mammary tissue, the roles played by gangliosides in breast cancer formation and metastasis are not clear.

Methods

To investigate the roles of gangliosides in breast tumor development, GM3 synthase was silenced in the highly metastatic 4T1 cells and over-expressed in the non-metastatic 67NR cells. The behavior of breast cancer cells was examined in vitro using migration assay, invasion assay, and soft agar assay. Tumor formation and metastasis in vivo were examined using a well established mouse mammary tumor model.

Results

GM3 synthase silencing in 4T1 cells significantly inhibited cell migration, invasion and anchorage-independent growth in vitro, and lung metastasis in vivo. In addition, over-expression of GM3 synthase in nonmetastatic 67NR cells significantly induced cell migration and anchorage-independent growth. Further studies indicated that activation of the phosphoinositide-3 kinase/Akt pathway, and consequently inhibition of nuclear factor of activated T cell (NFAT)1 expression, could be the mechanism underlying the suppression of breast cancer migration/invasion induced by GM3 synthase silencing.

Conclusion

Our findings indicate that GM3 synthase silencing suppressed lung metastasis in murine breast cancer cells. The molecular mechanism that underlies GM3 synthase mediated migration and invasion was inhibition of the phosphoinositide-3 kinase/Akt pathway. The findings suggest that GM3 synthase may be of value as a therapeutic target in breast cancer.     

Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)

Introduction

Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1, also known as EBP50 or NHERF) is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity (LOH) at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified.

Methods

To confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA (shRNA) to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses.

Results

We found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G₁-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level.

Conclusion

Our findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities.     

BMP-7 inhibits TGF-β-induced invasion of breast cancer cells through inhibition of integrin β(3) expression

Background

The transforming growth factor (TGF)-?? superfamily comprises cytokines such as TGF-?? and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-?? are associated with poor outcome, whereas inhibition of TGF-??-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.

Methods

In this study, we investigated the effect of BMP-7 on TGF-??-induced invasion in a 3 dimensional invasion assay.

Results

BMP-7 inhibited TGF-??-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-??-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-??-induced expression of integrin ??_v??₃ in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin ??₃ negatively affected TGF-??-induced invasion. On the other hand, overexpression of integrin ??₃ counteracted the inhibitory effect of BMP7 on TGF-??-induced invasion.

Conclusion

Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-??-induced expression of integrin ??₃.     

Adipose tissue derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor derived factors

Background

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs.

Methods

hASCs cultured under different conditions were quantified for the expression of alpha smooth muscle actin. ELISA was performed using the human TGFβ1, SDF-1α and CCL5 Quantikine Kit. The invasion potential of MDAMB231 cancer cells was evaluated using a Boyden chamber with filter inserts coated with Matrigel in 24-well dishes.

Results

We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g. expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell derived factor 1 (SDF-1) and CCL5.

Conclusion

Our data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.     

Epigenetic silencing of DSC3 is a common event in human breast cancer

Introduction

Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.

Methods

We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.

Results

DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.

Conclusion

These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.     

A novel taspine derivative,HMQ1611, suppresses adhesion,migration and invasion of ZR-75-30 human breast cancer cells

Background

Taspine was screened for the first time from Radix et Rhizoma leonticis (Hong Mao Qi in Chinese) using cell membrane chromatography in our laboratory. Its anticancer and antiangiogenic properties were demonstrated, and it could serve as a lead compound in anticancer agent development. Here, we investigated the role of one of the derivatives, HMQ1611, with increased activity and solubility, on the regulation of breast cancer cell ZR-75-30 adhesion, migration and invasion.

Methods

The effect of HMQ1611 on adhesion, invasion and migration of human breast cancer cells ZR-75-30 was examined. The migration and invasive potential of ZR-75-30 cells were examined by wound-healing assays and matrigel invasion chamber assays. The adhesion to type IV collagen and laminin were evaluated by MTT assay. The expression and proteinase activity of two matrix metalloproteinases (MMPs), matrix metalloproteinases 2 (MMP-2) and matrix metalloproteinases 9 (MMP-9), were analyzed by Western blot analysis and gelatin zymography, respectively.

Results

HMQ1611 effectively inhibited ZR-75-30 cell invasion and significantly suppressed adhesion to type IV collagen and laminin-coated substrate in a dose-dependent manner. Western blot and gelatin zymography analysis showed that HMQ1611 significantly inhibited the expression and secretion of MMP-2 and MMP-9 in ZR-75-30 cells. Additionally, treatment of ZR-75-30 cells with HMQ1611 downregulated the expression of MMP-2 and MMP-9.

Conclusions

HMQ1611 had potential to suppress the adhesion, migration and invasion of ZR-75-30 cancer cells, and it could serve as a potential novel therapeutic candidate for the treatment of metastatic breast cancer.     

EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells

Introduction

Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model.

Methods

SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice.

Results

The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo.

Conclusions

Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.     

Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis

Introduction

Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an antiinflammatory, antithrombosis, and antioxidant agent, and more recently, for its protective function in the pathogenesis of carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in inhibiting migration and invasion of breast cancer cells and to elucidate the underlying molecular mechanisms.

Methods

Clonogenicity and three-dimensional colony-formation assays were used to examine breast cancer cell growth with honokiol treatment. The effect of honokiol on invasion and migration of breast cancer cells was evaluated by using Matrigel invasion, scratch-migration, spheroid-migration, and electric cell-substrate impedance sensing (ECIS)-based migration assays. Western blot and immunofluorescence analysis were used to examine activation of the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) axis. Isogenic LKB1-knockdown breast cancer cell line pairs were developed. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of honokiol was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Finally, mouse xenografts, immunohistochemical and Western blot analysis of tumors were used.

Results

Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity, as evidenced by increased phosphorylation of the downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). By using AMPK-null and AMPK-WT (MEFs), we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor-suppressor LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and, more important, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increases in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors.

Conclusions

Taken together, these data provide the first in vitro and in vivo evidence of the integral role of the LKB1-AMPK axis in honokiol-mediated inhibition of the invasion and migration of breast cancer cells. In conclusion, honokiol treatment could potentially be a rational therapeutic strategy for breast carcinoma.     

Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo

Introduction

Adenine nucleotide translocator (ANT) 2 is highly expressed in proliferative cells, and ANT2 induction in cancer cells is known to be directly associated with glycolytic metabolisms and carcinogenesis. In addition, ANT2 repression results in the growth arrest of human cells, implying that ANT2 is a candidate for cancer therapy based on molecular targeting.

Methods

We utilized an ANT2-specific RNA interference approach to inhibit ANT2 expression for evaluating its antitumor effect in vitro and in vivo. Specifically, to investigate the therapeutic potential of ANT2 repression, we used a DNA vector-based RNA interference approach by expressing shRNA to knockdown ANT2 in breast cancer cell lines overexpressing ANT2.

Results

ANT2 shRNA treatment in breast cancer cell line MDA-MB-231 repressed cell growth as well as proliferation. In addition, cell cycle arrest, ATP depletion and apoptotic cell death characterized by the potential disruption of mitochondrial membrane were observed from the ANT2 shRNA-treated breast cancer cells. Apoptotic breast cancer cells transfected with ANT2 shRNA also induced a cytotoxic bystander effect that generates necrotic cell death to the neighboring cells. The intracellular levels of TNFα and TNF-receptor I were increased in ANT2 shRNA transfected cells and the bystander effect was partly blocked by anti-TNFα antibody. Ultimately, ANT2 shRNA effectively inhibited tumor growth in vivo.

Conclusion

These results suggest that vector-based ANT2 RNA interference could be an efficient molecular therapeutic method for breast cancer with high expression of ANT2.     

CXCR4 activation maintains a stem cell population in tamoxifen-resistant breast cancer cells through AhR signalling

Background:

Tamoxifen is commonly used for breast cancer therapy. However, tamoxifen resistance is an important clinical problem. Continuous treatment with conventional therapy may contribute to cancer progression in recurring cancers through the accumulation of drug-resistant cancer progenitors.

Methods:

To investigate signalling mechanisms important for the maintenance and viability of drug-resistant cancer progenitors, we used microarray analysis, PCR array for genes involved in cancer drug resistance and metabolism, flow cytometry, soft agar colony formation assay, in vivo tumourigenicity assay and immunohistochemical analysis using tamoxifen-sensitive and tamoxifen-resistant breast cancer MCF7 cells.

Results:

Downregulation of CXCR4 signalling by small molecule antagonist AMD3100 specifically inhibits growth of progenitor cell population in MCF7(TAM-R) cells both in vitro and in vivo. Microarray analysis revealed aryl hydrocarbon receptor (AhR) signalling as one of the top networks that is differentially regulated in MCF7(TAM-R) and MCF7 xenograft tumours treated with AMD3100. Further, small molecule antagonists of AhR signalling specifically inhibit the progenitor population in MCF7(TAM-R) cells and growth of MCF7(TAM-R) xenografts in vivo.

Conclusion:

The chemokine receptor CXCR4 maintains a cancer progenitor population in tamoxifen-resistant MCF7 cells through AhR signalling and could be a putative target for the treatment of tamoxifen-resistant breast cancers.     

Genes responsive to both oxidant stress and loss of estrogen receptor function identify a poor prognosis group of estrogen receptor positive primary breast cancers

Introduction

Oxidative stress can modify estrogen receptor (ER) structure and function, including induction of progesterone receptor (PR), altering the biology and clinical behavior of endocrine responsive (ER-positive) breast cancer.

Methods

To investigate the impact of oxidative stress on estrogen/ER-regulated gene expression, RNA was extracted from ER-positive/PR-positive MCF7 breast cancer cells after 72 hours of estrogen deprivation, small-interfering RNA knockdown of ER-α, short-term (8 hours) exposure to various oxidant stresses (diamide, hydrogen peroxide, and menadione), or simultaneous ER-α knockdown and oxidant stress. RNA samples were analyzed by high-throughput expression microarray (Affymetrix), and significance analysis of microarrays was used to define gene signatures responsive to estrogen/ER regulation and oxidative stress. To explore the association of these signatures with breast cancer biology, microarray data were analyzed from 394 ER-positive primary human breast cancers pooled from three independent studies. In particular, an oxidant-sensitive estrogen/ER-responsive gene signature (Ox-E/ER) was correlated with breast cancer clinical parameters and disease-specific patient survival (DSS).

Results

From 891 estrogen/ER-regulated probes, a core set of 75 probes (62 unique genes) responsive to all three oxidants were selected (Ox-E/ER signature). Ingenuity pathway analysis of this signature highlighted networks involved in development, cancer, and cell motility, with intersecting nodes at growth factors (platelet-derived growth factor-BB, transforming growth factor-β), a proinflammatory cytokine (tumor necrosis factor), and matrix metalloproteinase-2. Evaluation of the 394 ER-positive primary breast cancers demonstrated that Ox-E/ER index values correlated negatively with PR mRNA levels (r _p = -0.2; P = 0.00011) and positively with tumor grade (r _p = 0.2; P = 9.741 × e^-5), and were significantly higher in ER-positive/PR-negative versus ER-positive/PR-positive breast cancers (t-test, P = 0.0008). Regardless of PR status, the Ox-E/ER index associated with reduced DSS (n = 201; univariate Cox, P = 0.078) and, using the optimized cut-point, separated ER-positive cases into two significantly different DSS groups (log rank, P = 0.0009).

Conclusion

An oxidant-sensitive subset of estrogen/ER-responsive breast cancer genes linked to cell growth and invasion pathways was identified and associated with loss of PR and earlier disease-specific mortality, suggesting that oxidative stress contributes to the development of an aggressive subset of primary ER-positive breast cancers.     

Na+/H+ exchanger regulatory factor 1 inhibits platelet-derived growth factor signaling in breast cancer cells

Introduction

The gene encoding Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) is a putative tumor suppressor gene that harbors frequent loss of heterozygosity (LOH) and intragenic mutations in breast carcinoma. The exact biologic activity of NHERF1 in mammary glands, however, remains unclear. It was recently proposed that NHERF1 forms a ternary complex with platelet-derived growth factor receptor (PDGFR) and phosphatase and tensin homolog (PTEN), linking NHERF1 suppressor activity to PDGF-initiated phosphoinositide-3 kinase (PI3K)/PTEN signaling.

Methods

The effect of NHERF1 on the kinetics of PDGF-induced Akt activation was determined in cells with varied NHERF1 background. Levels of active Akt in mammary gland of NHERF1 knockout and wild-type mice were compared. We also examined how NHERF1 expression status affects cell sensitivity to PDGFR inhibitor. A plausible connection between NHERF1 and PTEN pathway was explored at the genetic level.

Results

We showed that NHERF1, through its PDZ-I domain, interacts directly with the carboxyl-terminal tail of PTEN. Knocking down NHERF1 expression in Zr75.1 cells markedly delayed the turnover of PDGF-induced phospho-Akt. Conversely, NHERF1 over-expression in MCF10A cells led to accelerated phospho-Akt degradation. The slowed decay of phospho-Akt that resulted from NHERF1 loss was evident in mouse embryonic fibroblasts isolated from NHERF1 knockout mice. In agreement with this, mammary gland tissues from these mice exhibited markedly elevated phospho-Akt. The responses of breast cancer cells to PDGFR inhibition were also altered by changes in NHERF1 expression level. Zr75.1 cells with NHERF1 knockdown were more resistant to STI-571-induced apoptosis than parental cells. Similarly, over-expression of NHERF1 rendered MCF10A cells more sensitive to STI-571. NHERF1-induced apoptotic response relies on an intact PTEN pathway; over-expression of NHERF1 in MCF10A cells with PTEN knockdown did not affect STI-571 sensitivity. It was found that NHERF1 LOH-positive breast cancer cells had reduced NHERF1 expression. Interestingly, these cells more frequently had wild-type PTEN or PI3KCA gene than the LOH-negative lines.

Conclusion

Our data indicate that the interaction of NHERF1 with PTEN counterbalances PI3K/Akt oncogenic signaling and may affect how cells respond to PDGFR inhibition in breast cancer. The dependence of NHERF1 responses on PTEN and genetic segregation of NHERF1 and PTEN (or PI3KCA) alterations suggest that NHERF1 is an active component of the PTEN pathway. Collectively, our study indicates that the biologic activity of NHERF1 in mammary gland is related to PTEN signaling.     

Human pituitary tumor-transforming gene 1 overexpression reinforces oncogene-induced senescence through CXCR2/p21 signaling in breast cancer cells

Introduction

hPTTG1 (human pituitary tumor-transforming gene 1) is an oncogene overexpressed in breast cancer and several other types of cancer. Increased hPTTG1 expression has been shown to be associated with poor patient outcomes in breast cancer. Although hPTTG1 overexpression plays important roles in promoting the proliferation, invasion, and metastasis of cancer cells, it also has been suggested to induce cellular senescence. Deciphering the mechanism by which hPTTG1 overexpression induces these contradictory actions in breast cancer cells is critical to our understanding of the role of hPTTG1 in breast cancer development.

Methods

MCF-10A and MCF-7 cells were used to identify the mechanism of hPTTG1-induced senescence. The interplay between hPTTG1 overexpression and chemokine C-X-C motif receptor 2 (CXCR2)/p21-dependent senescence in tumor growth and metastasis of MCF-7 cells was investigated by orthotopic transplantation of severe combined immunodeficiency (SCID) mice. Additionally, human invasive ductal carcinoma (IDC) tissue arrays were used to confirm the hPTTG1/CXCR2/p21 axis established in vitro.

Results

In this study, we investigated the mechanism of hPTTG1-induced senescence as well as its role in breast cancer progression and metastasis. Herein, we showed that hPTTG1 overexpression reinforced senescence through the CXCR2/p21 signaling. Furthermore, hPTTG1 overexpression activated NF-κB signaling to transactivate the expression of interleukin (IL)-8 and growth-regulated oncogene alpha (GROα) to execute CXCR2 signaling in MCF-7 cells. When CXCR2 expression was knocked down in hPTTG1-overexpressing MCF-7 cells, hPTTG1-induced senescence was abrogated by alleviating CXCR2-induced p21 expression. In a mouse model, CXCR2-mediated senescence limited hPTTG1-induced tumor growth and metastasis. Moreover, CXCR2 knockdown in hPTTG1-overexpressing MCF-7 tumors dramatically accelerated tumor growth and metastasis. Our in vitro and in vivo results demonstrated that hPTTG1 overexpression reinforces senescence through CXCR2 signaling, and the evasion of CXCR2/p21-dependent senescence was critical to hPTTG1 exerting its oncogenic potential. Interestingly, although CXCR2-dependent senescence restrained hPTTG1-induced tumor progression, when MCF-7 cells and hPTTG1-overexpressing MCF-7 cells were co-transplanted into the mammary fat pads of SCID mice, hPTTG1-overexpressing senescent cells created a metastasis-promoting microenvironment that promoted lung metastasis of the MCF-7 cells. Immunohistochemical analysis of human breast tumor samples also confirmed the importance of the hPTTG1/CXCR2 axis in promoting breast cancer metastasis.

Conclusions

Our findings provide novel molecular insights into hPTTG1-induced senescence and identify a novel mechanism by which hPTTG1 promotes metastasis by regulating the senescence-associated microenvironment.