Basal/HER2 breast carcinomas

High rates of inherent primary resistance to the humanized monoclonal antibody trastuzumab (Herceptin) are frequent among HER2 gene-amplified breast carcinomas in both metastatic and adjuvant settings. The clinical efficacy of trastuzumab is highly correlated with its ability to specifically and efficiently target HER2-driven populations of breast cancer stem cells (CSCs). Intriguingly, many of the possible mechanisms by which cancer cells escape trastuzumab involve many of the same biomarkers that have been implicated in the biology of CS-like tumor-initiating cells. In the traditional, one-way hierarchy of CSCs in which all cancer cells descend from special self-renewing CSCs, HER2-positive CSCs can occur solely by self-renewal. Therefore, by targeting CSC self-renewal and resistance, trastuzumab is expected to induce tumor shrinkage and further reduce breast cancer recurrence rates when used alongside traditional therapies. In a new, alternate model, more differentiated non-stem cancer cells can revert to trastuzumab-refractory, CS-like cells via the activation of intrinsic or microenvironmental paths-to-stemness, such as the epithelial-to-mesenchymal transition (EMT). Alternatively, stochastic transitions of trastuzumab-responsive CSCs might also give rise to non-CSC cellular states that lack major attributes of CSCs and, therefore, can remain “hidden” from trastuzumab activity. Here, we hypothesize that a better understanding of the CSC/non-CSC social structure within HER2-overexpressing breast carcinomas is critical for trastuzumab-based treatment decisions in the clinic. First, we decipher the biological significance of CSC features and the EMT on the molecular effects and efficacy of trastuzumab in HER2-positive breast cancer cells. Second, we reinterpret the genetic heterogeneity that differentiates trastuzumab-responders from non-responders in terms of CSC cellular states. Finally, we propose that novel predictive approaches aimed at better forecasting early tumor responses to trastuzumab should identify biological determinants that causally underlie the intrinsic flexibility of HER2-positive CSCs to “enter” into or “exit” from trastuzumab-sensitive states. An accurate integration of CSC cellular states and EMT-related biomarkers with the currently available breast cancer molecular taxonomy may significantly improve our ability to make a priori decisions about whether patients belonging to HER2 subtypes differentially enriched with a “mesenchymal transition signature” (e.g., luminal/HER2 vs. basal/HER2) would distinctly benefit from trastuzumab-based therapy ab initio.     

Lapatinib,a dual HER1/HER2 tyrosine kinase inhibitor,augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2‐driven cancer cell growth

The ultimate biological and clinical meaning of shed HER2 extracellular domain (ECD) has remained largely unclear until recently. Oversecretion of soluble HER2 ECD has been shown to inhibit growth of HER2‐overexpressing cancer cells by promoting HER2 ECD dimerization with HER transmembrane receptors thus impairing their cross‐tyrosine phosphorylation and decreasing their activation status. HER2‐targeted drugs capable to enhance the occurrence of basal HER2 ECD shedding but simultaneously preventing formation of truncated cell membrane‐bound HER2 intracellular fragment, which exhibits an undesirable constitutive kinase activity, might be extremely efficient at managing HER2‐positive cancer disease. The dual HER1/HER2 Tyrosine Kinase inhibitor lapatinib, which works intracellularly and directly targets the TK domain of HER2, drastically augments basal shedding of HER2 ECD to inhibit HER2‐driven cancer cell growth. Lapatinib treatment significantly augments the concentration of the inactive (unphosphorylated) form of HER2 protein at the tumor cell membrane and promotes an exacerbated HER2 ECD shedding to the extracellular milieu of HER2‐overexpressing cancer cells. Exacerbated sensitivity of trastuzumab‐resistant cancer cells, which contain nearly undetectable levels of soluble HER2 ECD when compared with trastuzumab‐sensitive parental cells to lapatinib‐induced cell growth inhibition, takes place when lapatinib treatment fully restores high levels of basal HER2 ECD shedding. The dramatic augmentation of HER2 ECD shedding that occurs upon treatment of with lapatinib is fully suppressed in lapatinib‐refractory HER2‐positive cells. These findings, altogether, may provide crucial insights concerning clinical studies aimed to accurately describe HER2 ECD as a potential predictor of response or resistance to the HER2‐targeted drugs trastuzumab and lapatinib. J. Cell. Physiol. 226: 52–57, 2010. © 2010 Wiley‐Liss, Inc.     

Reverse-phase protein microarray highlights HER2 signaling activation in immunohistochemistry/FISH/HER2-negative breast cancers

Evaluation of: Wulfkuhle JD, Berg D, Wolff C et al. Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping. Clin. Cancer Res. 18(23), 6426–6435 (2012).

Exhaustive characterization and mapping of pivotal molecules and downstream effectors deregulated in breast cancer is of fundamental clinical value to define the most effective therapy. Wulfkuhle et al. applied reverse-phase protein microarray, a highly sensitive immunoassay able to perform quantitative and multiplexed analysis of total and/or modified cellular proteins, to assess protein levels and activation/phosphorylation status of the HER family (EGFR, HER2, HER3) and downstream signaling molecules in HER2⁺ and HER2^- breast cancers. The research was performed using laser capture microdissected tumor epithelial cells from frozen samples and formalin-fixed paraffin embedded specimens, which were also analyzed by immunohistochemistry (IHC) and FISH. This study identified a subgroup of IHC/FISH/HER2^- patients with HER2 activation/phosphorylation levels comparable with those obtained from IHC/FISH/HER2⁺ tumors. HER2 signaling activation was independent from total HER2 expression and involved HER3 and EGFR activation. These findings indicate that molecular characterization by reverse-phase protein microarray of HER2 and its partners/effectors in the signaling cascade enables the identification of a subgroup of IHC/FISH/HER2^- patients showing HER2 signaling activation. These patients, currently excluded from targeted therapy administration, could potentially benefit from this and it could improve prognosis and survival.     

Model-Based Analysis of HER Activation in Cells Co-Expressing EGFR,HER2 and HER3

The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.     

Molecular dynamics simulations of asymmetric heterodimers of HER1/HER2 complexes

The family of human epidermal growth factor receptors (HER) is involved in tumor cell growth. Homodimerization and heterodimerization of the HER family are important for activation of these receptors. The structures of homodimer conformation are well characterized, while the structures of heterodimer conformations, especially between HER1 and HER2, are not completely understood. In this study, two models of possible asymmetric HER1/HER2 kinase domains were built. Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) methods were applied to examine the possibility of these two-heterodimer interactions. From our results, it could be concluded that the HER2 kinase domain prefers to serve as the receiver rather than the activator. Key binding residues of this dimer complex at N lobe of HER2 is ALA683 and at C lobe of HER1 are GLU914, GLU917, and ASP930. This study will be useful in allowing us to predict and be able to control activity of this enzyme in disease in the future.

Open image in new window

Graphical abstract A model of the asymmetric dimer of HER1–HER2 heterodimer with key intereaction residues in (a) HER1A and (b) HER2R by molecular dynamic simulation

     

Calmodulin binds HER2 and modulates HER2 signaling

Human epidermal growth factor receptor 2 (HER2), a member of the ErbB family of receptor tyrosine kinases, has defined roles in neoplastic transformation and tumor progression. Overexpression of HER2 is an adverse prognostic factor in several human neoplasms and, particularly in breast cancer, correlates strongly with a decrease in overall patient survival. HER2 stimulates breast tumorigenesis by forming protein-protein interactions with a diverse array of intracellular signaling molecules, and evidence suggests that manipulation of these associations holds therapeutic potential. To modulate specific HER2 interactions, the region(s) of HER2 to which each target binds must be accurately identified. Calmodulin (CaM), a ubiquitously expressed Ca²⁺ binding protein, interacts with multiple intracellular targets. Interestingly, CaM binds the juxtamembrane region of the epidermal growth factor receptor, a HER2 homolog. Here, we show that CaM interacts, in a Ca²⁺-regulated manner, with two distinct sites on the N-terminal portion of the HER2 intracellular domain. Deletion of residues 676-689 and 714-732 from HER2 prevented CaM-HER2 binding. Inhibition of CaM function or deletion of the CaM binding sites from HER2 significantly decreased both HER2 phosphorylation and HER2-stimulated cell growth. Collectively, these data suggest that inhibition of CaM-HER2 interaction may represent a rational therapeutic strategy for the treatment of patients with breast cancer. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3.

Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor-derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG-induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3-kinase in transfected 293 and mammary carcinoma-derived MCF-7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG-dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG-induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.     

Dysregulation of HER2/HER3 signaling axis in Epstein-Barr virus-infected breast carcinoma cells

The role of Epstein-Barr virus (EBV) in the pathogenesis of breast cancer has been of long-standing interest to the field. Breast epithelial cells can be infected by EBV through direct contact with EBV-bearing lymphoblastoid cells, and EBV infection has recently been shown to confer breast cancer cells an increased resistance to chemotherapeutic drugs. In this study, we established EBV-infected breast cancer MCF7 and BT474 cells and demonstrated that EBV infection promotes tumorigenic activity of breast cancer cells. Firstly, we showed that the EBV-infected MCF7-A and BT474-A cells exhibited increased anchorage-independent growth in soft agar. The increased colony formation capacity in soft agar was associated with increased expression and activation of HER2/HER3 signaling cascades, as evidenced by the findings that the treatment of HER2 antibody trastuzumab (Herceptin), phosphatidylinositol 3-kinase inhibitor, or MEK inhibitor completely abolished the tumorigenic capacity. In the EBV-infected breast cancer cells, the expression of EBV latency genes including EBNA1, EBER1, and BARF0 was detected. We next showed that BARF0 alone was sufficient to efficiently up-regulate HER2/HER3 expression and promoted tumorigenic activity in MCF7 and BT474 cells by the use of both overexpression and small interfering RNA knock-down. Collectively, we demonstrated that EBV-encoded BARF0 promotes the tumorigenic activity of breast cancer cells through activation of HER2/HER3 signaling cascades.     

Targeting HER2

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A”-DTPA yielded a chelate:protein ratio of 3.4±0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian, and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models, and the highest tumor %ID/g (51.18±13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85±273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts, was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected, but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.     

Houttuyninum, an active constituent of Chinese herbal medicine, inhibits phosphorylation of HER2/neu receptor tyrosine kinase and the tumor growth of HER2/neu-overexpressing cancer cells

AimsThe overexpression of HER2/neu receptor plays a key role in tumorigenesis and tumor progression. Small molecules targeting HER2/neu have therapeutic value in cancers that overexpress HER2. In this present study, the effect of houttuyninum, a component in the Chinese herbal medicine Houttuynia cordata Thunb, on HER2/neu tyrosine phosphorylation and its in vivo antitumour activity was investigated.Main methodsThe phosphorylation and expression of proteins were determined by Western blot analysis. The MTT assay was employed to examine the inhibition of cell proliferation in vitro. Xenografts were established in nude mice for evaluating the antitumour activity of houttuyninum in vivo.Key findingsHouttuyninum inhibited phosphorylation of HER2 in a dose-dependent manner with an IC50 of 5.52 μg/ml without reducing HER2/neu protein expression in MDA-MB-453 cells. Houttuyninum also inhibited the activation of ERK1/2 and AKT, downstream molecules in the HER2/neu-mediated signal transduction pathway. In contrast, tyrosine phosphorylation of EGFR was unaffected when the concentration of houttuyninum was increased to 40 μg/ml in both A431 cells and MDA-MB-468 cells. Additionally, houttuyninum preferentially inhibited the growth of MDA-MB-453 cells that overexpressed HER2/neu; the MDA-MB-468 cells that overexpress EGFR remained unaffected. Administration of houttuyninum in vivo resulted in a significant reduction of phosphorylated HER2 levels and in tumor volumes of the BT474 and N87 xenografts, which both overexpress HER2/neu.SignificanceOur findings showed that houttuyninum can inhibit the HER2/neu signalling pathway and the tumor growth of cancer cells that overexpress HER2/neu. This drug may provide therapeutic value in the treatment of cancers that involve overexpression of HER2/neu.     

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.     

Fluorescent silica nanospheres for digital counting bioassay of the breast cancer marker HER2/neu [correction of HER2/nue

This paper describes the use of fluorescent silica nanospheres as luminescent signal amplifiers in biological assays based on digital counting of individual particles instead of measuring averaged fluorescence intensity. We recently described a simple method to prepare highly fluorescent mono-dispersed silica nanospheres that avoids microemulsion formulations and the use of surfactants. Modification of the St?ber method was used successfully to prepare fluorescent silica spheres with the inorganic dye dichlorotris(1,10-phenanathroline)ruthenium (II) hydrate encapsulated during the condensation of tetraethylorthosilicate in ethanol and dye aqueous mixtures. Modifications in the ammonia and water content in the reaction mixture resulted in mono-dispersed silica spheres of 65, 440 and 800 nm in diameter. The dye-encapsulating particles emit intense red luminescence when excited at 460 nm. We observed an increased photostability and longer fluorescence lifetime in our particles that we attributed to increased protection of the encapsulated dye molecules from molecular oxygen. The newly prepared fluorescent silica particles were easily modified using trialkoxysilane reagents for covalent conjugation of anti-HER2/neu. We demonstrated the utility of the fluorescent nanospheres to detect the cancer marker HER2/neu in a glass slide based assay. The assay was shown to be simple but highly sensitive with a limit of detection approaching 1 ng/mL and a linear range between 1 ng/mL and 10 microg/mL of HER2/neu.     

Correlation of HER2, p95HER2 and HER3 Expression and Treatment Outcome of Lapatinib plus Capecitabine in her2-Positive Metastatic Breast Cancer

Background

Lapatinib plus capecitabine is an effective treatment option for trastuzumab-refractory HER2-positive metastatic breast cancer. We have investigated the correlation between quantitative measures of HER2, p95HER2, and HER3 and treatment outcomes using lapatinib and capecitabine.

Methods

Total HER2 (H2T), p95HER2 (p95), and total HER3 (H3T) expression were quantified in formalin-fixed paraffin-embedded samples using the VeraTag assays. Patients received lapatinib and capecitabine treatment following trastuzumab failure according to the Lapatinib Expanded Access Program. The association between the protein expression levels and clinical outcomes was analyzed.

Results

A total of 52 patients were evaluable. H2T level was significantly higher in responders (median 93.49 in partial response, 47.66 in stable disease, and 17.27 in progressive disease; p = 0.020). Longer time-to-progression (TTP) was observed in patients with high H2T [p = 0.018, median 5.2 months in high (>14.95) vs. 1.8 in low (<14.95)] and high H3T [p = 0.017, median 5.0 months in high (>0.605) vs. 2.2 in low (<0.605)]. Patients having both high H2T and high H3T had significantly longer TTP [adjusted hazard ratio (HR) 0.38 (95% CI 0.20–0.73), p = 0.004] and overall survival [adjusted HR 0.46 (95% CI 0.24–0.89), p = 0.020]. No significant association between p95 and response or survival was observed.

Conclusions

These data suggest a correlation between high HER2 and high HER3 expression and treatment outcome, while no significant difference was observed between clinical outcome and p95 expression level in this cohort of HER2-positive, trastuzumab-refractory metastatic breast cancer patients treated with lapatinib and capecitabine.     

CTLs directed against HER2 specifically cross-react with HER3 and HER4

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2(369-377) epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-gamma treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2(369-377)-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3(356-364) and HER4(361-369) were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.     

Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.     

Overexpression of HER2/neu oncoprotein in cytologic specimens

OBJECTIVE: To assess the rate of HER2/neu overexpression in cytologic specimens by immunocytochemistry (ICC) and compare these results in matched surgical specimens by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), when available. STUDY DESIGN: All cytologic specimens processed for HER2/neu evaluation by ICC (72 cases) and available corresponding histologic specimens (16 cases) were retrieved from our files. ICC was applied to previously Papanicolaou stained, routine fine needle aspirations specimens (64 cases) and cytocentrifuged, alcohol-fixed, fluid specimens (8 cases). FISH was performed on 6 histologic specimens. RESULTS: Overexpression of HER2/neu was seen in 7/22 breast cancers (31.8%), 3/18 pulmonary adenocarcinomas (16.6%), 2/5 colorectal adenocarcinomas (40%), 1/2 adenocarcinomas of the biliary system (50%), 1/3 thyroid papillary carcinomas (33.3%) and 1/3 prostate adenocarcinomas (33.3%). Sixteen cases had IHC in matched histologic specimens: 14 (87.5%) cases were concordant (11 negative and 3 positive in both specimens), 1 case was negative in the cytologic specimen and positive in the histologic specimen (with no amplification by FISH), and 1 case was positive in the cytologic specimen and negative in the histologic specimen (not informative by FISH). CONCLUSION: Our data suggest that overexpression of HER2/neu oncoprotein can be successfully detected in routine cytologic specimens, providing a simple, fast and cost-effective method of selecting patients for specific treatment.     

Dmp1α Inhibits HER2/neu-Induced Mammary Tumorigenesis

Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21^Cip1, markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression.