Metastasis remains the leading cause of cancer morbidity and mortality. ErbB2, a metastasis-promoting oncoprotein, is overexpressed in 50–60% of noninvasive ductal carcinoma in situ (DCIS). However, only 25% of invasive breast cancer (IBC) overexpress ErbB2, indicating that ErbB2 alone is not sufficient to drive metastasis and additional risk factors are necessary for the progression of ErbB2-overexpressing DCIS to IBC. A recent study published in Cancer Cell identified 14-3-3ξ as a risk factor aiding the transition of ErbB2-overexpressing DCIS into IBC. Furthermore, the study elucidated molecular mechanisms by which ErbB2 and 14-3-3ξ co-overexpression drives metastasis. Namely, ErbB2 promotes cell motility and migration via the activation of Src, while 14-3-3ξ induces epithelial-mesenchymal transition by activating TGFβ pathway to reduce cell adhesion. On the other hand, two studies recently published in British Journal of Cancer and Oncogene provide mechanistic insight into how ErbB signalling is transduced via Src, focal adhesion kinase and Ste20-like kinase to regulate focal adhesion turnover and modulate cell motility and migration. Taken together, these studies reveal that metastasis engages a variety of players that must show team spirit to win the game of spreading.
ErbB2; 14-3-3ξ; metastasis; invasive breast cancer; epithelial-mesenchymal transition; focal adhesion; cell motility; cell adhesion
The processes that control the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain poorly understood. Epidermal growth factor receptor 2 (ErbB2) over expression is common in DCIS, as is disruption of the retinoblastoma tumor suppressor (RB) pathway. Here we examined the cooperative impact of ErbB2 and RB deregulation on facets of disease progression. Our studies demonstrate that RB deficiency altered the expression of key molecules needed for proper cellular organization and epithelial cell-cell adhesion as part of a program related to the epithelial to mesenchymal transition (EMT). An increase in the invasive potential of ErbB2 over expressing cells was observed upon RB depletion. Furthermore, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared to DCIS-like lesions developing from RB-proficient cells. Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner. Lastly, in a cohort of DCIS cases, we show that while elevated levels of ErbB2 are associated with increased risk of a subsequent DCIS recurrence, it is not associated with progression to invasive disease. In contrast, RB loss in ErbB2 positive DCIS cases was associated with increased risk for invasive breast cancer. Taken together, these data demonstrate a key role for the RB-pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease.
Tumor Suppressor; Epithelial to Mesenchymal Transition; Cyclin Dependent Kinase; Cell Cycle; E2F; CDK4/6; PD-0332991
Expression of c-erbB3 protein was investigated in 104 primary breast carcinomas comprising nine comedo ductal carcinoma in situ (DCIS), 91 invasive ductal carcinomas and four invasive lobular carcinomas using two monoclonal antibodies, RTJ1 and RTJ2. Of the 91 invasive ductal carcinomas, seven contained the comedo DCIS component adjacent to the invasive component. An immunohistochemical technique was used to evaluate the association between expression of c-erbB3 and clinical parameters and tumour markers such as epidermal growth factor receptor (EGFR), c-erbB2, cathepsin-D and p53 in archival formalin-fixed paraffin-embedded tumour tissues. Our results indicated that RTJ1 and RTJ2 gave identical staining patterns and concordant results. It was found that the overexpression of c-erbB3 protein was observed in 67% (6/9) of comedo DCIS, 52% (44/84) of invasive ductal carcinomas, 71% (5/7) of carcinomas containing both the in situ and invasive lesions and 25% (1/4) of invasive lobular carcinomas. A significant relationship (P < 0.05) was observed between strong immunoreactivity of c-erbB3 protein and histological grade, EGFR and cathepsin-D, but not with expression of c-erbB2, p53, oestrogen receptor status, lymph node metastases or age of patient. However, we noted that a high percentage of oestrogen receptor-negative tumours (59%), lymph node-positive tumours (63%) and c-erbB2 (63%) were strongly positive for c-erbB3 protein. We have also documented that a high percentage of EGFR (67%), c-erbB2 (67%), p53 (75%) and cathepsin-D-positive DCIS (60%) were strongly positive for c-erbB3. These observations suggest that overexpression of c-erbB3 protein could play an important role in tumour progression from non-invasive to invasive and, also, that it may have the potential to be used as a marker for poor prognosis of breast cancer.
ErbB2 is frequently highly expressed in premalignant breast cancers, including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it contributes to progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here, we show that clinically relevant doses of ionizing radiation (IR)-induce cellular invasion of ErbB2-expressing breast cancer cells, as well as MCF10A cells overexpressing ErbB2. ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR nor do MCF10A cells overexpressing epidermal growth factor receptor. ErbB2 becomes phosphorylated at tyrosine 877 in a dose- and time- dependent manner following exposure to X-rays, and activates downstream signaling cascades including PI3K/Akt. Inhibition of these pathways, as well as inhibition of reactive oxygen species (ROS) with antioxidants, prevents IR-induced invasion. Activation of ErbB2-dependent signaling results in upregulation of the forkhead family transcription factor, FoxM1, and its transcriptional targets, including matrix metalloproteinase 2 (MMP2). Inhibition of FoxM1 by RNA interference prevented induction of invasion by IR, and overexpression of FoxM1 in MCF10A cells was sufficient to promote IR-induced invasion. Moreover, we found that 14-3-3ζ is also upregulated by IR in cancer cells in a ROS-dependent manner, is required for IR-induced invasion in ErbB2-positive breast cancer cells and together with FoxM1 is sufficient for invasion in ErbB2-negative breast cancer cells. Thus, our data show that IR-mediated activation of ErbB2 and induction of 14-3-3ζ collaborate to regulate FoxM1 and promote invasion of breast cancer cells and furthermore, may serve as therapeutic targets to enhance radiosensitivity of breast cancers.
ErbB2; FoxM1; 14-3-3ζ; invasion; reactive oxygen species
Increasing evidence suggests that HER2-amplified breast cancer cells use HER3/ErbB3 to drive therapeutic resistance to HER2 inhibitors. However, the role of ErbB3 in the earliest events of breast epithelial transformation remains unknown. Using mouse mammary specific models of Cre-mediated ErbB3 ablation, we show that ErbB3 loss prevents the progressive transformation of HER2-overexpressing mammary epithelium. Decreased proliferation and increased apoptosis were seen in MMTV-HER2 and MMTV-Neu mammary glands lacking ErbB3, thus inhibiting premalignant HER2-induced hyperplasia. Using a transgenic model in which HER2 and Cre are expressed from a single polycistronic transcript, we showed that palpable tumor penetrance decreased from 93.3% to 6.7% upon ErbB3 ablation. Penetrance of ductal carcinomas in situ was also decreased. In addition, loss of ErbB3 impaired Akt and p44/42 phosphorylation in preneoplastic HER2-overexpressing mammary glands and in tumors, decreased growth of preexisting HER2-overexpressing tumors, and improved tumor response to the HER2 tyrosine kinase inhibitor lapatinib. These events were rescued by reexpression of ErbB3, but were only partially rescued by ErbB36F, an ErbB3 mutant harboring six tyrosine-to-phenylalanine mutations that block its interaction with phosphatidyl inositol 3-kinase. Taken together, our findings suggest that ErbB3 promotes HER2-induced changes in the breast epithelium before, during, and after tumor formation. These results may have important translational implications for the treatment and prevention of HER2-amplified breast tumors through ErbB3 inhibition.
The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in ~30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2α inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2α can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2α phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis.
We tested whether ErbB2 signaling influenced eIF2α signaling and whether enhanced phosphorylation of the latter affected ErbB2-deregulated mammary acinar development. We obtained stable MCF10A cells overexpressing wild-type (Wt) Neu/ErbB2 or a constitutively active (CA) variant via retroviral delivery or mammary tumor cells from MMTV-Neu tumors. Western blotting, RT-PCR and confocal microscopy were used to analyze the effects of ErbB2 activation on eIF2α signaling and the effect of the GADD34-PP1C inhibitor salubrinal. Wt- and MMTV-Neu cells formed aberrant acini structures resembling DCIS, while CA-ErbB2 overexpression induced invasive lesions. In these structures we found that CA-ErbB2 but not the Wt variant significantly down-regulated the pro-apoptotic gene CHOP. This occurred without apparent modulation of basal phosphorylation of PERK and eIF2α or induction of its downstream target ATF4. However, inhibition of eIF2α dephosphorylation with salubrinal was sufficient to inhibit Wt- and CA-ErbB2- as well as MMTV-Neu-induced deregulation of acinar growth. This was linked to enhanced CHOP expression, inhibition of proliferation, induction of apoptosis and luminal clearing in Wt-ErbB2 and to inhibition of cyclin D1 levels and subsequent proliferation in CA-ErbB2 cells.
Depending on the strength of ErbB2 signaling there is a differential regulation of CHOP and eIF2α phosphorylation. ErbB2 uncouples in basal conditions eIF2α phosphorylation from CHOP induction. However, this signal was restored by salubrinal treatment in Wt-ErbB2 expressing MCF10A cells as these DCIS-like structures underwent luminal clearing. In CA-ErbB2 structures apoptosis is not induced by salubrinal and instead a state of quiescence with reduced proliferation was achieved. Treatments that stabilize P-eIF2α levels may be effective in treating ErbB2 positive cancers without severely disrupting normal tissue function and structure.
AIMS: To assess c-erbB-2 immunostaining in relation to morphological type of in situ and invasive breast carcinoma. METHODS: Formalin fixed, wax embedded archival tissue was used. Invasive carcinomas comprised 50 infiltrating ductal (NOS); seven medullary, 10 tubular, 15 mucinous and 24 classic invasive lobular. In situ carcinomas comprised 48 ductal (DCIS) and 10 cases of lobular (LCIS). The antibodies used were pAB1 (polyclonal) which stains cell lines that over express the c-erbB-2 oncogene, and ICR 12 (monoclonal) which stains sections of breast carcinoma known to show c-erbB-2 amplification. RESULTS: Immunostaining consistent with c-erbB-2 overexpression was found in 10 out of 50 cases of infiltrating ductal carcinoma (NOS), one of 24 infiltrating lobular carcinomas and one of seven medullary carcinomas only. Seventy per cent of ICR 12 positive cases of infiltrating ductal carcinoma also had extratumoral DCIS. Forty six per cent of pure DCIS lesions also showed strong membrane staining for c-erbB-2 protein, confined to large cell types. CONCLUSIONS: Immunostaining for c-erb B-2 oncoprotein occurs mainly in large cell DCIS and infiltrating ductal carcinoma NOS, especially those with an extratumoral DCIS component. There is a low incidence in other types of breast cancer, including those associated with a better prognosis. Different biological mechanisms may be responsible for histologically distinct types of breast carcinoma.
The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.
Heregulin (HRG; also known as neuregulin) is a ligand for ErbB3. One of its isotypes, HRG-β1, binds to ErbB3 and forms heterodimers with other ErbB family members, thereby enhancing the proliferation and tumorigenesis of breast cancer cells. HRG stimulation may contribute to the progression of epithelial–mesenchymal transition (EMT) and tumor metastasis in breast cancer. Majority of studies regarding EMT has been concentrated on TGF-β signaling. Therefore, we investigated whether the HRG-β1 and ErbB3 activate Smad2 signaling during process of EMT in breast cancer cells.
The SK-BR-3 and MCF7 breast cancer cell lines were used. The expressions of phospho-Smad2 and EMT markers were observed by western blotting and immunofluorescence assays after treatment with HRG-β1. The cell motility and invasiveness were determined by wound healing and matrigel invasion assays. Smad2 and ErbB3 small interfering RNA (siRNA) transfections were performed to assess the involvement of ErbB3 and Smad2 in HRG-β1-induced EMT.
HRG-β1 induced EMT through activation of Smad2. The expression of E-cadherin was decreased after HRG-β1 treatment, while the expressions of Snail, vimentin, and fibronectin were increased. The HRG-β1-induced expressions of Snail, vimentin, and fibronectin, and nuclear colocalization of phospho-Smad2 and Snail were inhibited by pretreatment with a PI3k inhibitor, LY294002, or two phospho-Smad2 inhibitors, PD169316 or SB203580 and cancer cell migration by HRG-β1 was inhibited. Knockdown of Smad2 by siRNA transfection suppressed the expressions of Snail and fibronectin in response to HRG-β1 stimulation and knockdown of ErbB3 suppressed the expressions of phospho-Smad2, Snail, and fibronectin induced by HRG-β1, whereas E-cadherin was increased compared with control siRNA-transfected cells. Knockdown of ErbB3 and Smad2 also decreased SK-BR-3 and MCF7 cell invasion.
Our data suggest that HRG-β1 and ErbB3 induce EMT, cancer cell migration and invasion through the PI3k/Akt-phospho-Smad2-Snail signaling pathway in SK-BR-3 and MCF7 breast cancer cells.
Heregulin; Transforming growth factor-β (TGF-β); Smad2; EMT; Breast cancer; ErbB3; Small interfering RNA (siRNA)
BACKGROUND: The overexpression or amplification of tumor suppressor and proto-oncogenes are important factors in the progression of breast cancer. Recent attention has focused on the cyclin genes, whose involvement in signal transduction pathways regulate cell cycle progression. The amplification of the cyclins D1 and D3 genes usually leads to loss of normal growth control and is thought to play an important growth regulatory role in tumor development and progression. In this report, we investigate the association of altered cyclin expression with other prognostic indicators (histological grade, lymph node status, estrogen receptor, p53, and c-erbB-2) in the progression of human breast cancer. MATERIALS AND METHODS: Surgical tumor specimens were obtained from 16 breast tubular ductal, and invasive ductal carcinomas and grafted onto gnotobiotic nude (nu/nu) mice. The expression diversity and distribution of the localization of the protein products of the c-erbB-2, cyclins D1 and D3, p53, and estrogen receptor were characterized immunohistochemically and the results in the original tumor (T0) were compared with those in the tumors that developed in nude mice (T1) xenografts. RESULTS: The T0 tumors exhibited a diversity of cellular morphology in the tumor matrix and diversity in expression of these proteins. These specific changes were also preserved in the T1 tumors. Whereas 67% of the T1 tumors exhibited high numbers of estrogen receptorpositive nuclei, only 50% of these tumors grew when grafted onto nude mice. The histological grade (14/15 were G2 to G3) and metastatic malignancy in the lymph nodes (10/15) did not appear to be related to tumor growth in the nude mouse. There was no relationship between those tumors which exhibited high percentages of c-erbB-2- and p53-positive cells and growth in nude mice. A strong association (p < 0.001) was observed between the overexpression of cyclin D1 transcripts in the T0 tumors and the continued growth of the T1 tumors in nude mice. In the T1 tumors, both cyclins D1 and D3, estrogen receptor, and p53 were observed in 49% to 86% of the cells of the T1 tumors examined; the number of cells expressing c-erbB-2 protein varied widely in these tumors. CONCLUSIONS: The results indicate that the tumor matrix exhibits a diversity in the level of phenotypic expression of genes involved in cellular growth of breast tumors in both the T0 or T1 host environment. Changes in cyclin activity appear to correlate with the vigorous level of breast tumor growth and progression.
Well over a quarter of human breast cancers are ErbB2-driven and constitute a distinct subtype with substantially poorer prognosis. Yet, there are substantial gaps in our understanding of how ErbB2 tyrosine kinase activity unleashes a coordinated program of cellular and extracellular alterations that culminate in aggressive breast cancers. Cellular models that exhibit ErbB2 kinase dependency and can induce metastatic breast cancer in immune competent hosts are likely to help bridge this gap.
Materials and Methods:
Here, we derived and characterized a cell line model obtained from a transgenic ErbB2/Neu-driven mouse mammary adenocarcinoma.
The MPPS1 cell line produces metastatic breast cancers when implanted in the mammary fat pads of immune-compromised as well as syngeneic immune-competent hosts. MPPS1 cells maintain high ErbB2 overexpression when propagated in DFCI-1 or related media, and their growth is ErbB2-dependent, as demonstrated by concentration-dependent inhibition of proliferation with the ErbB kinase inhibitor Lapatinib. When grown in 3-dimensional (3-D) culture on Matrigel, MPPS1 cells predominantly form large irregular cystic and solid structures. Remarkably, low concentrations of Lapatinib led to a switch to regular acinar growth on Matrigel. Immunofluorescence staining of control vs. Lapatinib-treated acini for markers of epithelial polarity revealed that inhibition of ErbB2 signaling led to rapid resumption of normal mammary epithelium-like cell polarity.
The strict dependence of the MPPS1 cell system on ErbB2 signals for proliferation and alterations in cell polarity should allow its use to dissect ErbB2 kinase-dependent signaling pathways that promote loss of cell polarity, a key component of the epithelial mesenchymal transition and aggressiveness of ErbB2-driven breast cancers.
3-D Matrigel; EMT; ErbB2/Her2/Neu; lapatinib; mouse models; syngeneic xenografts
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
microRNA (miRNA) are short, noncoding RNA that negatively regulate gene expression and may play a causal role in invasive breast cancer. Since many genetic aberrations of invasive disease are detectable in early stages, we hypothesized that miRNA expression dysregulation and the predicted changes in gene expression might also be found in early breast neoplasias.
Expression profiling of 365 miRNA by real-time quantitative polymerase chain reaction assay was combined with laser capture microdissection to obtain an epithelium-specific miRNA expression signature of normal breast epithelium from reduction mammoplasty (RM) (n = 9) and of paired samples of histologically normal epithelium (HN) and ductal carcinoma in situ (DCIS) (n = 16). To determine how miRNA may control the expression of codysregulated mRNA, we also performed gene expression microarray analysis in the same paired HN and DCIS samples and integrated this with miRNA target prediction. We further validated several target pairs by modulating the expression levels of miRNA in MCF7 cells and measured the expression of target mRNA and proteins.
Thirty-five miRNA were aberrantly expressed between RM, HN and DCIS. Twenty-nine miRNA and 420 mRNA were aberrantly expressed between HN and DCIS. Combining these two data sets with miRNA target prediction, we identified two established target pairs (miR-195:CCND1 and miR-21:NFIB) and tested several novel miRNA:mRNA target pairs. Overexpression of the putative tumor suppressor miR-125b, which is underexpressed in DCIS, repressed the expression of MEMO1, which is required for ErbB2-driven cell motility (also a target of miR-125b), and NRIP1/RIP140, which modulates the transcriptional activity of the estrogen receptor. Knockdown of the putative oncogenic miRNA miR-182 and miR-183, both highly overexpressed in DCIS, increased the expression of chromobox homolog 7 (CBX7) (which regulates E-cadherin expression), DOK4, NMT2 and EGR1. Augmentation of CBX7 by knockdown of miR-182 expression, in turn, positively regulated the expression of E-cadherin, a key protein involved in maintaining normal epithelial cell morphology, which is commonly lost during neoplastic progression.
These data provide the first miRNA expression profile of normal breast epithelium and of preinvasive breast carcinoma. Further, we demonstrate that altered miRNA expression can modulate gene expression changes that characterize these early cancers. We conclude that miRNA dysregulation likely plays a substantial role in early breast cancer development.
Pathways involved in DCIS stem and progenitor signalling are poorly understood yet are critical to understand DCIS biology and to develop new therapies. Notch and ErbB1/2 receptor signalling cross talk has been demonstrated in invasive breast cancer, but their role in DCIS stem and progenitor cells has not been investigated. We have utilised 2 DCIS cell lines, MCF10DCIS.com (ErbB2-normal) and SUM225 (ErbB2-overexpressing) and 7 human primary DCIS samples were cultured in 3D matrigel and as mammospheres in the presence, absence or combination of the Notch inhibitor, DAPT, and ErbB1/2 inhibitors, lapatinib or gefitinib. Western blotting was applied to assess downstream signalling. In this study we demonstrate that DAPT reduced acini size and mammosphere formation in MCF10DCIS.com whereas there was no effect in SUM225. Lapatinb reduced acini size and mammosphere formation in SUM225, whereas mammosphere formation and Notch1 activity were increased in MCF10DCIS.com. Combined DAPT/lapatinib treatment was more effective at reducing acini size in both DCIS cell lines. Mammosphere formation in cell lines and human primary DCIS was reduced further by DAPT/lapatinib or DAPT/gefitinib regardless of ErbB2 receptor status. Our pre-clinical human models of DCIS demonstrate that Notch and ErbB1/2 both play a role in DCIS acini growth and stem cell activity. We report for the first time that cross talk between the two pathways in DCIS occurs regardless of ErbB2 receptor status and inhibition of Notch and ErbB1/2 was more efficacious than either alone. These data provide further understanding of DCIS biology and suggest treatment strategies combining Notch and ErbB1/2 inhibitors should be investigated regardless of ErbB2 receptor status.
We reported that the class I HDAC inhibitor entinostat induced apoptosis in erbB2-overexpressing breast cancer cells via downregulation of erbB2 and erbB3. Here, we study the molecular mechanism by which entinostat dual-targets erbB2/erbB3. Treatment with entinostat had no effect on erbB2/erbB3 mRNA, suggesting a transcription-independent mechanism. Entinostat decreased endogenous but not exogenous erbB2/erbB3, indicating it did not alter their protein stability. We hypothesized that entinostat might inhibit erbB2/erbB3 protein translation via specific miRNAs. Indeed, entinostat significantly upregulated miR-125a, miR-125b, and miR-205, that have been reported to target erbB2 and/or erbB3. Specific inhibitors were then used to determine whether these miRNAs had a causal role in entinostat-induced downregulation of erbB2/erbB3 and apoptosis. Transfection with a single inhibitor dramatically abrogated entinostat induction of miR-125a, miR-125b, or miR-205; however, none of the inhibitors blocked entinostat action on erbB2/erbB3. In contrast, co-transfection with two inhibitors not only reduced their corresponding miRNAs, but also significantly abrogated entinostat-mediated reduction of erbB2/erbB3. Moreover, simultaneous inhibition of two, but not one miRNA significantly attenuated entinostat-induced apoptosis. Interestingly, although the other HDAC inhibitors, such as SAHA and panobinostat, exhibited activity as potent as entinostat to induce growth inhibition and apoptosis in erbB2-overexpressing breast cancer cells, they had no significant effects on the three miRNAs. Instead, both SAHA- and panobinostat-decreased erbB2/erbB3 expression correlated with the reduction of their mRNA levels. Collectively, we demonstrate that entinostat specifically induces expression of miR-125a, miR-125b, and miR-205, which act in concert to downregulate erbB2/erbB3 in breast cancer cells. Our data suggest that epigenetic regulation via miRNA-dependent or -independent mechanisms may represent a novel approach to treat breast cancer patients with erbB2-overexpressing tumors.
entinostat; HDAC; miRNA; erbB2; erbB3; breast cancer
Advances in high-throughput genomic-scanning have expanded the repertory of genetic variations in DNA sequences encoding ErbB tyrosine kinase receptors in humans, including single nucleotide polymorphisms (SNPs), polymorphic repetitive elements, microsatellite variations, small-scale insertions and deletions. The ErbB family members: EGFR, ErbB2, ErbB3, and ErbB4 receptors are established as drivers of many aspects of tumor initiation and progression to metastasis. This knowledge has provided rationales for the development of an arsenal of anti-ErbB therapeutics, ranging from small molecule kinase inhibitors to monoclonal antibodies. Anti-ErbB agents are becoming the cornerstone therapeutics for the management of cancers that overexpress hyperactive variants of ErbB receptors, in particular ErbB2-positive breast cancer and non-small cell lung carcinomas. However, their clinical benefit has been limited to a subset of patients due to a wide heterogeneity in drug response despite the expression of the ErbB targets, attributed to intrinsic (primary) and to acquired (secondary) resistance. Somatic mutations in ErbB tyrosine kinase domains have been extensively investigated in preclinical and clinical setting as determinants for either high sensitivity or resistance to anti-ErbB therapeutics. In contrast, only scant information is available on the impact of SNPs, which are widespread in genes encoding ErbB receptors, on receptor structure and activity, and their predictive values for drug susceptibility. This review aims to briefly update polymorphic variations in genes encoding ErbB receptors based on recent advances in deep sequencing technologies, and to address challenging issues for a better understanding of the functional impact of single versus combined SNPs in ErbB genes to receptor topology, receptor-drug interaction, and drug susceptibility. The potential of exploiting SNPs in the era of stratified targeted therapeutics is discussed.
ErbB receptors; cancer; SNPs; anti-ErbB therapeutics; drug response; resistance
LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration, which was reported to be overexpressed in 8–12 % of human breast cancers and thought to be exclusively located in cytoplasm.
In the present work we analyzed the cellular and histological expression pattern of LASP-1 and its involvement in biological behavior of human breast cancer through correlation with standard clinicopathological parameters and expression of c-erbB2 (HER-2/neu), estrogen- (ER) and progesterone-receptors (PR). For this purpose immunohistochemical staining intensity and percentage of stained cells were semi-quantitatively rated to define a LASP-1 immunoreactive score (LASP-1-IRS). LASP-1-IRS was determined in 83 cases of invasive ductal breast carcinomas, 25 ductal carcinomas in situ (DCIS) and 18 fibroadenomas. Cellular LASP-1 distribution and expression pattern was visualized by immunofluorescence and confocal microscopy and assessed through separate Western blots of nuclear and cytosol preparations of BT-20, MCF-7, MDA-MB231, and ZR-75/1 breast cancer cells.
Statistical analysis revealed that the resulting LASP-1-IRS was significantly higher in invasive carcinomas compared to fibroadenomas (p = 0.0176). Strong cytoplasmatic expression of LASP-1 was detected in 55.4 % of the invasive carcinomas, which correlated significantly with nuclear LASP-1-positivity (p = 0.0014), increased tumor size (p = 0.0159) and rate of nodal-positivity (p = 0.0066). However, levels of LASP-1 expression did not correlate with average age at time point of diagnosis, histological tumor grading, c-erbB2-, ER- or PR-expression.
Increased nuclear localization and cytosolic expression of LASP-1 was found in breast cancer with higher tumor stage as well as in rapidly proliferating epidermal basal cells. Confocal microscopy and separate Western blots of cytosolic and nuclear preparations confirmed nuclear localization of LASP-1.
The current data provide evidence that LASP-1 is not exclusively a cytosolic protein, but is also detectable within the nucleus. Increased expression of LASP-1 in vivo is present in breast carcinomas with higher tumor stage and therefore may be related with worse prognosis concerning patients' overall survival.
ErbB2, a member of the epidermal growth factor receptor (EGFR) family, is overexpressed in 20% to 30% of human breast cancer cases and forms oncogenic signalling complexes when dimerised to ErbB3 or other EGFR family members.
We crossed mouse mammary tumour virus (MMTV)-myr-Akt1 transgenic mice (which express constitutively active Akt1 in the mammary gland) with MMTV-c-ErbB2 transgenic mice to evaluate the role of Akt1 activation in ErbB2-induced mammary carcinoma using immunoblot analysis, magnetic resonance spectroscopy and histological analyses.
Bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 mice develop mammary tumours twice as fast as MMTV-c-ErbB2 mice. The bitransgenic tumours were less organised, had more mitotic figures and fewer apoptotic cells. However, many bitransgenic tumours displayed areas of extensive necrosis compared with tumours from MMTV-c-ErbB2 mice. The two tumour types demonstrate dramatically different expression and activation of EGFR family members, as well as different metabolic profiles. c-ErbB2 tumours demonstrate overexpression of EGFR, ErbB2, ErbB3 and ErbB4, and activation/phosphorylation of both ErbB2 and ErbB3, underscoring the importance of the entire EGFR family in ErbB2-induced tumourigenesis. Tumours from bitransgenic mice overexpress the myr-Akt1 and ErbB2 transgenes, but there was dramatically less overexpression and phosphorylation of ErbB3, diminished phosphorylation of ErbB2, decreased level of EGFR protein and undetectable ErbB4 protein. There was also an observable attenuation in a subset of tyrosine-phosphorylated secondary signalling molecules in the bitransgenic tumours compared with c-ErbB2 tumours, but Erk was activated/phosphorylated in both tumour types. Finally, the bitransgenic tumours were metabolically more active as indicated by increased glucose transporter 1 (GLUT1) expression, elevated lactate production and decreased intracellular glucose (suggesting increased glycolysis).
Expression of activated Akt1 in MMTV-c-ErbB2 mice accelerates tumourigenesis with a reduced requirement for signalling through the EGFR family, as well as a reduced requirement for a subset of downstream signaling molecules with a metabolic shift in the tumours from bitransgenic mice. The reduction in signalling downstream of ErbB2 when Akt is activated suggest a possible mechanism by which tumour cells can become resistant to ErbB2-targeted therapies, necessitating therapies that target oncogenic signalling events downstream of ErbB2.
Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression. ErbB2 targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. In the present study, using a clonal population of GW583340 (lapatinib analog, ErbB1/2 inhibitor)-resistant IBC cells, we identified overexpression of an anti-apoptotic protein, XIAP, in acquired resistance to GW583340 in both ErbB2 overexpressing SUM190 and ErbB1 activated SUM149 cell lines derived from primary IBC tumors. A marked decrease in p-ErbB2, p-ErbB1, and downstream signaling was evident in the GW583340-resistant cells (rSUM190 and rSUM149) similar to parental counterparts treated with the drug, suggesting the primary mechanism of action of GW583340 was not compromised in resistant cells. However, rSUM190 and rSUM149 cells growing in GW583340 had significant XIAP overexpression and resistance to GW583340-mediated apoptosis. Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. XIAP downregulation in rSUM190 and rSUM149 cells using a small molecule inhibitor (embelin), which abrogates the XIAP/procaspase 9 interaction, resulted in decreased viability, demonstrating that XIAP is required for survival of cells with acquired resistance to GW583340. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to ErbB2-targeting agents.
IRES; embelin; survivin; FOXO3a; p-AKT
Elevated expression of erbB3 receptor has been reported to induce resistance to therapeutic agents, including trastuzumab in erbB2-overexpressing breast cancer. Our recent studies indicate that erbB3 interacts with both erbB2 and IGF-1 receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121/SAR256212, a fully human anti-erbB3 antibody (Ab), against two erbB2-overexpressing breast cancer cell lines resistant to trastuzumab.
MTS-based proliferation assays were used to determine cell viability upon treatment of trastuzumab and/or MM-121/SAR256212. Cell cycle progression was examined by flow cytometric analysis. Western blot analyses were performed to determine the expression and activation of proteins. Tumor xenografts were established by inoculation of the trastuzumab-resistant BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with trastuzumab and/or MM-121/SAR256212 via i.p injection to determine the Abs’ antitumor activity. Immunohistochemical analyses were carried out to study the Abs’ inhibitory effects on tumor cell proliferation and induction of apoptosis in vivo.
MM-121 significantly enhanced trastuzumab-induced growth inhibition in two sensitive and two resistant breast cancer cell lines. MM-121 in combination with trastuzumab resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and Akt (P-Akt) in the in vitro studies. MM-121 combined with trastuzumab did not induce apoptosis in the trastuzumab-resistant cell lines under our cell culture condition, rather induced cell cycle G1 arrest mainly associated with the upregulation of p27kip1. Interestingly, in the tumor xenograft model established from the trastuzumab-resistant cells, MM-121 in combination with trastuzumab as compared to either agent alone dramatically inhibited tumor growth correlated with a significant reduction of Ki67 staining and increase of cleaved caspase-3 in the tumor tissues.
The combination of MM-121 and trastuzumab not only inhibits erbB2-overexpressing breast cancer cell proliferation, but also promotes the otherwise trastuzumab-resistant cells undergoing apoptosis in an in vivo xenografts model. Thus, MM-121 exhibits potent antitumor activity when combined with trastuzumab under the studied conditions. Our data suggest that further studies regarding the suitability of MM-121 for treatment of breast cancer patients whose tumors overexpress erbB2 and become resistant to trastuzumab may be warranted.
MM-121; SAR256212; erbB3; erbB2; Trastuzumab resistance; Breast cancer
The expression of p53 protein, oestrogen receptor protein, epidermal growth factor receptor (EGFR) and overexpression of the c-erbB-2 oncoprotein was examined in a series of 149 primary symptomatic breast carcinomas. Expression of p53 was present in 62 of 146 cases (42.5%) of the invasive carcinoma and one of three cases (33.3%) of ductal carcinoma in situ (DCIS) examined. Statistical associations of tumour oestrogen receptor positivity and lack of p53 protein expression, chi 2 = 19.78 (d.f. = 1), P less than 0.001, positive tumour p53 status and poor tumour grade; chi 2 = 14.1 (d.f. = 2), P less than 0.001, EGFR expression chi 2 = 7.07, (d.f. = 1), P less than 0.01 and tumour c-erbB-2 protein overexpression; chi 2 = 4.61 (d.f. = 1), P = 0.032 were identified. Expression of p53 is rare in invasive lobular carcinoma of classical type (8.3% of cases examined) in contrast to other common types of mammary carcinoma. Non-significant trends of p53 protein expression and increased regional tumour recurrence; chi 2 = 3.20 (d.f. = 1), P = 0.074 and also poorer patient survival; chi 2 = 3.76 (d.f. = 1), P = 0.053 were identified. p53 protein expression is a common event in human breast cancer and is present in both DCIS and invasive mammary carcinoma. Abnormal expression of p53 protein is a feature of both in situ and invasive breast carcinoma, implying that the abnormal p53 protein expression may be implicated in the early stages of mammary carcinoma progression.
The ErbB2/3 heterodimer plays a critical role in breast cancer progression and in the development of endocrine resistance. EBP1, an ErbB3 binding protein, inhibits HRG-stimulated breast cancer growth, decreases ErbB2 protein levels and contributes to tamoxifen sensitivity. We report here that ectopic expression of EBP1 in Estrogen Receptor (ER) positive breast cancers that express ErbB2 at both high and low levels decreased ErbB2 protein levels. ErbB2 protein expression was also increased in mammary glands of Ebp1 knock out mice. To define the mechanism of ErbB2 down regulation, we examined the effects of EBP1 on ErbB2 mRNA levels, transcription of the ErbB2 gene and ErbB2 protein stability. We found that ectopic expression of EBP1 decreased steady state levels of endogenous ErbB2 mRNA in all cell lines tested. EBP1 overexpression decreased the activity of an ErbB2 promoter reporter in cells which overxpress ErbB2. However, reporter activity was unchanged or increased in cells which express low endogenous levels of ErbB2. We also found that ectopic expression of EBP1 accelerated ErbB2 protein degradation and enhanced ErbB2 ubiquitination in cells which express both low and high levels of ErbB2. Treatment with proteasome inhibitors prevented this decrease in ErbB2 protein levels. Ablation of EBP1 expression led to tamoxifen resistance that was abrogated by inhibition of ErbB2 activity. These results suggest that EBP1 inhibits expression of ErbB2 protein levels by multiple mechanisms and that EBP1’s effects on tamoxifen sensitivity are mediated in part by its ability to modulate ErbB2 levels.
EBP1; ErbB2; tamoxifen; breast cancer
Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype.
CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (Â± siRNA-mediated CD44 suppression) or MCF7 cells (Â± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA.
TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration.
These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance.
Tamoxifen-resistance; CD44; erbB; Hyaluronan; Heregulin
Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has been shown to play a key role in invasion and metastasis of breast carcinoma cells. However, very little is known about its role in normal tissue homeostasis. Here, we investigated the effects of LOXL2 expression in normal mammary epithelial cells to gain insight into how LOXL2 mediates cancer progression.
LOXL2 was expressed in MCF10A normal human mammary epithelial cells. The 3D acinar morphogenesis of these cells was assessed, as well as the ability of the cells to form branching structures on extracellular matrix (ECM)-coated surfaces. Transwell-invasion assays were used to assess the invasive properties of the cells. Clinically relevant inhibitors of ErbB2, lapatinib and Herceptin (traztuzumab), were used to investigate the role of ErbB2 signaling in this model. A retrospective study on a previously published breast cancer patient dataset was carried out by using Disease Specific Genomic Analysis (DSGA) to investigate the correlation of LOXL2 mRNA expression level with metastasis and survival of ErbB2-positive breast cancer patients.
Fluorescence staining of the acini revealed increased proliferation, decreased apoptosis, and disrupted polarity, leading to abnormal lumen formation in response to LOXL2 expression in MCF10A cells. When plated onto ECM, the LOXL2-expressing cells formed branching structures and displayed increased invasion. We noted that LOXL2 induced ErbB2 activation through reactive oxygen species (ROS) production, and ErbB2 inhibition by using Herceptin or lapatinib abrogated the effects of LOXL2 on MCF10A cells. Finally, we found LOXL2 expression to be correlated with decreased overall survival and metastasis-free survival in breast cancer patients with ErbB2-positive tumors.
These findings suggest that LOXL2 expression in normal epithelial cells can induce abnormal changes that resemble oncogenic transformation and cancer progression, and that these effects are driven by LOXL2-mediated activation of ErbB2. LOXL2 may also be a beneficial marker for breast cancer patients that could benefit most from anti-ErbB2 therapy.
Hsp90 (heat shock protein90) is a chaperone protein essential for preserving and regulating the function of various cellular proteins. Elevated Hsp90 expression seems to be a trait of breast cancer and may be an integral part of the coping mechanisms that cancer cells exhibit vis-à-vis stress. This manuscript tries to examine the immunohistochemical expression of Hsp90 all along the continuum of breast ductal lesions encompassing ductal hyperplasia without atypia (DHWithoutA), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC).
Tissue specimens were taken from 30 patients with DHWithoutA, 31 patients with ADH, 51 with DCIS and 51 with IDC. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment the percentage of positive cells and the intensity were separately analyzed. Subsequently, the Allred score was calculated. Post hoc analysis on the correlations between Hsp90 Allred score and possible predictors (grade, nodal status, tumor size, ER Allred score, PR Allred score, c-erbB-2 status and triple negative status) was conducted in IDC.
Hsp90 exhibited mainly cytoplasmic immunoreactivity. Hsp90 Allred score exhibited an increasing trend along the continuum of breast ductal lesions (Spearman's rho = 0.169, p = 0.031). Compared to the adjacent normal ducts and lobules, no statistically significant differences were noted in DHwithoutA, ADH and DCIS. Hsp90 expression (intensity, positive cells, Allred score) was higher in IDC, compared to the adjacent normal tissue. Higher Hsp90 expression was observed in grade 2/3 IDCs (borderline association) and tumors of larger size. At the univariable analysis, higher Hsp90 expression was associated with higher ER Allred score, PR Allred score and c-erbB-2 positivity in IDC. Triple-negative IDCs exhibited significantly lower Hsp90 expression. The multivariable logistic regression model revealed that between the three markers, solely ER Allred score and c-erbB-2 positivity were independently associated with higher Hsp90 expression in IDC.
The above point to significant variability in Hsp90 expression with significant implications upon the effectiveness and limitations of anti-Hsp90 drugs.