Serum Thyrotropin Concentrations are not Predictive of Aggressive Breast Cancer Biology in Euthyroid Individuals

Objective: The potential influence of hypothyroidism on breast cancer remains incompletely understood. The objective of this study was to investigate the relationship between serum thyrotropin (thyroid-stimulating hormone, TSH) concentration and markers of aggressive breast cancer biology as defined by receptor expression profile, tumor grade, and American Joint Committee on Cancer (AJCC) stage characteristics.Methods: This was a retrospective cohort study of patients from 2002 to 2014. All breast cancer patients who had complete receptor (estrogen receptor, ER; progesterone receptor, PR; and human epidermal growth factor receptor 2, Her2/neu) and prediagnosis serum TSH data (n = 437) were included. All patients had 1 of 6 receptor profiles: ER+ PR+ Her2/neu-, ER+ PR- Her2/neu-, ER+ PR+ Her2/neu+, ER+ PR- Her2/neu+, ER- PR- Her2/neu+, or ER- PR- Her2/neu-. Log-transformed serum TSH concentrations were analyzed using multinomial and logistic regressions to identify potential relationships with markers of breast cancer aggressiveness.Results: Increasing serum TSH concentration was associated with a lower probability of having the receptor expression profile ER+ PR+ Her2/neu+ compared to patients with the ER+ PR+ Her2/neu- profile (odds ratio [OR] = 0.52, P = .0045). No significant associations between other receptor expression profiles and serum TSH concentration were found. All time-weighted and unweighted median serum TSH concentrations were within normal limits. No significant associations between serum TSH concentration and tumor grade, overall AJCC stage, tumor size (T), lymph node positivity (N), or presence of metastasis (M) were observed.Conclusions: Serum TSH was not associated with markers of breast cancer aggressiveness in our cohort.Abbreviations: AJCC = American Joint Committee on Cancer ER = estrogen receptor Her2/neu = human epidermal growth factor receptor 2 M = metastasis N = lymph node positivity OR = odds ratio PR = progesterone receptor T = tumor size TSH = thyroid stimulating hormone UCLA = University of California Los Angeles     

Her-2/neu oncogene expression in Puerto Rican females with breast cancer.

Her-2/neu belongs to the family of tyrosine kinase transmembrane proteins whose overexpression has been associated with a poor prognosis in patients with breast cancer. The product of this proto-oncogene is overexpressed in 25-30% of human breast cancer and is the target of selective immunotherapy. Concerned about the ethnic differences on the expression of this oncogene, we have evaluated 143 consecutive specimens of primary breast cancer diagnosed in San Pablo Hospital, Puerto Rico. The specimens were analyzed for Her-2/neu expression using immunohistochemistry assays (Hercept test). We have related the expression of hormone receptor status, percent cells in S phase, DNA ploidy, tumor size, nodal status and menopausal state with the Her2/neu expression. Out of 143 specimens, 28 overexpressed the Her-2/neu (19.6%). Of the Her/2 negative 30/114 (26%) were estrogen receptor negative as compared to 9/27 (33%) (p = 0.464). The degree of aneuploidy was abnormal in 25/104 (24%) in the Her-2/neu negative vs 11/27 (41%) p = 0.083. The percent cell in DNA synthesis was high in 16/77 (21%) in Her-2/neu negative vs 4/15 (27%) p = 0.613. The tumor size was greater than 2 cm in 35/106 (33%) in Her-2/neu negative vs 9/23 (39%) p = 0.575. In the progesterone specimens negative for Her2, 44/114 (39%) were Her2/neu negative vs 15/27 (56%) p = 0.108. No differences were seen regarding menopausal status, age and nuclear grading. A trend favoring abnormal aneuploidy in Her2/neu positive was seen. Nodal involvement was significantly associated with Her2/neu overexpression. (p = 0.037). Although the incidence of Her2 overexpression found in this database was somewhat lower than the one reported in the literature, this might also be due to the small cohort examined or to the technique utilized.     

Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered.     

Targeting HER2/neu with a fully human IgE to harness the allergic reaction against cancer cells

Breast and ovarian cancer are two of the leading causes of cancer deaths among women in the United States. Overexpression of the HER2/neu oncoprotein has been reported in patients affected with breast and ovarian cancers, and is associated with poor prognosis. To develop a novel targeted therapy for HER2/neu expressing tumors, we have constructed a fully human IgE with the variable regions of the scFv C6MH3-B1 specific for HER2/neu. This antibody was expressed in murine myeloma cells and was properly assembled and secreted. The Fc region of this antibody triggers in vitro degranulation of rat basophilic cells expressing human FcεRI (RBL SX-38) in the presence of murine mammary carcinoma cells that express human HER2/neu (D2F2/E2), but not the shed (soluble) antigen (ECD(HER2)) alone. This IgE is also capable of inducing passive cutaneous anaphylaxis in a human FcεRIα transgenic mouse model, in the presence of a cross-linking antibody, but not in the presence of soluble ECD(HER2). Additionally, IgE enhances antigen presentation in human dendritic cells and facilitates cross-priming, suggesting that the antibody is able to stimulate a secondary T-cell anti-tumor response. Furthermore, we show that this IgE significantly prolongs survival of human FcεRIα transgenic mice bearing D2F2/E2 tumors. We also report that the anti-HER2/neu IgE is well tolerated in a preliminary study conducted in Macaca fascicularis (cynomolgus) monkeys. In summary, our results suggest that this IgE should be further explored as a potential therapeutic against HER2/neu overexpressing tumors, such as breast and ovarian cancers.     

Rapid adaptation of a recombinant vesicular stomatitis virus to a targeted cell line

Vesicular stomatitis virus (VSV) is being developed for cancer therapy. We created a recombinant replicating VSV (rrVSV) that preferentially infected Her2/neu-expressing breast cancer cells. This rrVSV did not express the native VSV-G glycoprotein (gp). Instead, it expressed a chimeric Sindbis gp which included a single-chain antibody (SCA) directed to the human Her2/neu receptor. The virus infected mouse mammary carcinoma cells (D2F2/E2) expressing Her2/neu 23-fold better than the parent cells (D2F2). However, viral growth in cultured D2F2/E2 cells was curtailed after several cycles, and viral yield was very poor at 2 x 10(4) infectious doses (ID)/ml. We performed in vitro serial passage in D2F2/E2 cells to evolve a virus with improved growth that could be used for preclinical therapy trials in mice. Fifteen passes generated an adapted virus that progressed through multiple cycles in cultured D2F2/E2 cells until all cells were infected and had a viral yield of 1 x 10(8) ID/ml. Sequencing of the entire viral genomes found only 2 mutations in the adapted virus. Both mutations occurred in the gp gene segment coding for the SCA. An additional N-glycosylation site was created by one of the mutations. The adapted virus showed higher density of gp on the viral envelope, improved infectivity, much greater stability, higher burst size, and decreased induction of cellular interferon. The specificity for cells expressing the Her2/neu receptor was unchanged. These studies demonstrate that serial passage can be used to rapidly evolve a VSV genome encoding an improved chimeric glycoprotein.     

T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment

A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.     

Abstracts for a conference on trace elements in diet,nutrition, and health: essentiality and toxicity

Overexpression of HER2/neu is associated with drug resistance and poor outcome in breast cancer. Solamargine (SM), a glycoalkaloid purified from the herb Solanum incanum, exhibits HER2/neu gene modulation of HER2/neu high-expressing human breast cancer cell line ZR-75-1. SM downregulation of HER2/neu gene expression was determined by RT-PCR and Southern hybridization. Additionally, the membrane-bound HER2/neu receptor in highly HER2/neu-expressing breast cancer cells was determined by radioimmunoassay, immunocytochemistry, fluorescent immunocytochemistry, and flow cytometry. SM significantly decreased the number of HER2/neu receptors on the cell membrane. Methotrexate (MTX), 5-florouracil (5-Fu), and cisplatin (CDDP) are commonly used for breast carcinoma treatment in clinics; however, patients with HER2/neu overexpression exhibit resistance to these anticancer drugs. Notably, combination of MTX, 5-Fu, and CDDP with SM individually increased the susceptibility of breast cancer cells to these chemotherapeutic agents. Experimental results indicated that downregulation of HER2/neu by SM might be an effective strategy for enhancing drug susceptibility of breast cancer cells expressing high levels of HER2/neu.     

An active murine-human chimeric Fab antibody derived from Escherichia coli, potential therapy against over-expressing VEGFR2 solid tumors

Many recombinant murine monoclonal antibodies (mAbs) were studied under pre-clinical or clinical development and became one of the most prolific drug classes in oncology. Vascular endothelial growth factors receptor 2 (VEGFR2) has been implicated to play an important role in tumors. We have established a murine anti-VEGFR2 mAb. To reduce the shortcoming of the mAb, a murine–human chimeric Fab (cFab) named FA8H1 was constructed with gene engineering techniques and expressed as a soluble and functional protein in Escherichia coli Top10F′. Several immunological methods were used to characterize the cFab, including ELISA, affinity and kinetics assay, IP, IF, FACS, and IHC. The results illuminated that cFab maintained the specificity for the VEGFR2 antigen. The active cFab also effectively identified VEGFR2 over-expressing cells in a number of archived human cancer tissues, compared to its parental antibody. The FA8H1 provided the basis for potential therapy research against over-expressing VEGFR2 human solid tumors.     

Rational therapeutic intervention in cancer: kinases as drug targets.

Landmark clinical studies of new drugs developed to target specific forms of cancer were reported in 2001. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her-2/neu positive metastatic breast cancer, when combined with chemotherapy. STI-571, a small molecule inhibitor of the Bcr-Abl, c-kit and platelet derived growth factor receptor tyrosine kinases, produced dramatic clinical responses in patients with Bcr-Abl positive chronic myeloid leukemia and c-kit positive gastrointestinal stromal tumors. These examples have galvanized the cancer research community to extend kinase-inhibitor therapy to other cancers.     

Tyrosine kinases. New target of anticancer therapy

Recently, clinical studies of new drugs development to target specific forms of cancer were reported. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her2/neu positive metastatic breast cancer. STI571, a small molecule inhibitor of the BCR/ABL, c-Kit and platelet derived growth factor receptor tyrosine kinase, produced pronounced clinical responses in patients with BCR/ABL positive chronic myeloid leukemia and c-Kit positive gastrointestial stromal tumors. In order to consider the use of the inhibitor of tyrosine kinases activity as anticancer drug, their mechanisms of the oncogenic activation and their impact on tumor transformation should be studied. The treatment with tyrosine kinase inhibitors such as STI571 or herceptin was a spectacular clinical success which stimulated research on the structure and function of both kinases and their inhibitors.     

Comparison of adjuvant activity of N- and C-terminal domain of gp96 in a Her2-positive breast cancer model

It has been frequently reported that gp96 acts as a strong biologic adjuvant. Some studies have even investigated adjuvant activity of the gp96 C- or N-terminal domain. The controversy surrounding adjuvant activity of gp96 terminal domains prompted us to compare adjuvant activity of gp96 C- or N-terminal domain toward Her2/neu, as DNA vaccine in a Her2/neu-positive breast cancer model. To do so, mice were immunized with DNA vaccine consisting of transmembrane and extracellular domain (TM + ECD) of rat Her2/neu alone or fused to N- or C-terminal domain of gp96. Treatment with Her2/neu fused to N-terminal domain of gp96 resulted in tumor progression, compared to the groups vaccinated with pCT/Her2 or pHer2. Immunological examination revealed that treatment with Her2/neu fused to N-terminal domain of gp96 led to significantly lower survival rates, higher interferon-γ secretion, and induced infiltration of CD4⁺/CD8⁺ cells to the tumor site. However, it could not induce cytotoxic T lymphocyte activity, did not decrease regulatory T cell percentage at the tumor site, and eventually led to tumor progression. Our results reveal that gp96 N-terminal domain does not have adjuvant activity toward Her2/neu. It is also proposed that adjuvant activity and the resultant immune response of gp96 terminal domains may be directed by the antigen applied.     

Her4 and Her2/neu Tyrosine Kinase Domains Dimerize and Activate in a Reconstituted in Vitro System

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues ⁹⁵²ENI⁹⁵⁴ to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

Levels of specific peptide-HLA class I complex predicts tumor cell susceptibility to CTL killing

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed, suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag, HLA-A2 molecule, and Her2(369)-A2 complex expression. However, compared with untreated cells, cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells, the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further, these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells.     

Biological studies and potential therapeutic applications of monoclonal antibodies and small molecules reactive with theneu/c-erbB-2 protein

The overexpression of the growth factor receptor p185 ^neu/c-erbB-2 has been observed in a number of human adenocarcinomas and is mechanistically linked to neoplastic growth. Monoclonal antibodies raised against extracellular domains of the p185 ^neu/c-erbB-2 receptor oncoprotein have been utilized to inhibit the pathway ofneu-induced tumor development. Our laboratory has demonstrated a direct effect of anti-p185 ^neu/c-erbB2 antibodies which requires receptor ligation. This induced aggregation causes the downmodulation of cell-surface expression and eventual degradation of p185 ^neu/c-erbB-2 protein. In cells transformed by theneu oncogene, the result of antibody-induced p185 ^neu/c-erbB-2 receptor modulation is the reversion of the malignant pheno-type. We are exploiting the direct efficacy of this monoclonal antibody by developing small molecules (peptides and organic mimietics) based on anti-p185 ^neu/c-erbB-2 antibody structure that can mediate similar receptor binding and biological effects.     

Co-expression of steroid hormone receptors (estrogen receptor alpha and/or progesterone receptors) and Her2/neu (c-erbB-2) in breast cancer: clinical outcome following tamoxifen-based adjuvant therapy

In breast cancer patients, the expression of steroid hormone receptors (HR:ERalpha/PR) appears inversely correlated with Her2/neu (not all reports agree on this negative correlation). Moreover, some but not all studies suggest that HR+/Her2/neu+ patients have a poor response to endocrine therapy, making this special group a matter of debate. In this prospective study we have analyzed the clinical outcome of our HR+/Her2/neu+ patients (n=51) selected from 516 consecutive stages I-II cases, with a follow-up 5-10 years (mean 7.3), treated with standard adjuvant therapy with tamoxifen (TAM) (TAM alone or TAM after chemotherapy). This group was compared with the HR+/Her-2/neu- patients (n=129) treated also with TAM. The tumor biopsies were studied by immunohistochemistry. We found that the association HR+/Her2/neu- was 2.5 times higher than the association HR+/Her2/neu+ (25% versus 9.9%, respectively). Our study also showed that the disease free survival (DFS) of the patients co-expressing HR and Her2/neu was significantly lower than those expressing HR but lacking of Her2/neu (p<0.001). A similar result was obtained when the overall survival (OS) was evaluated (p=0.001). All of these patients received hormone therapy with TAM, alone or after chemotherapy. When the analysis was performed in the patients treated with TAM alone, again the expression of Her-2/neu had a negative impact on both the DFS and the OS (p<0.05).     

Adjuvant activity of GP96 C-terminal domain towards Her2/neu DNA vaccine is fusion direction-dependent

The Her2 is one of tumor-associated antigens (TAA), regarded as an ideal target of immunotherapy. DNA encoding full-length or truncated rat Her2/neu have shown protective and therapeutics potentials against Her2/neu-expressing mammary tumors. However, the efficacy of active vaccination is limited since Her2 is a self-tolerated antigen. Hence, new strategies are required to enhance both the quality and quantity of the immune response against Her2-expressing tumors. Many studies have used Her2/neu gene with cytokine or other molecules involved in regulation of immune response to enhance the potency of Her2/neu DNA vaccines. Some studies fused adjuvant gene to C-terminal domain of Her2/neu gene, while others fused the adjuvant gene N-terminally to Her2/neu gene, but no comparison on how direction of fusion could affect efficiency of DNA vaccine has ever been made. Based on previous reports demonstrating potent adjuvant activity of gp96 C-terminal domain, we chose it as adjuvant. The aim of this study was to investigate if direction of fusion could affect adjuvant activity of gp96 C-terminal domain or potency of Her2/neu DNA vaccination. To do so, we fused C-terminal domain of gp96 to downstream or C-terminal end of transmembrane and extracellular domain (TM+ECD) of rat Her2/neu and resultant immune response to DNA vaccination was evaluated. The results were compared with that of N-terminally fusion of gp96 C-terminal domain to TM+ECD of rat Her2/neu. Our results revealed that adjuvant activity of gp96 C-terminal domain is enhanced when fused N-terminally to TM+ECD of rat Her2/neu. It suggests that adjuvant activity of gp96 C-terminal domain towards Her2/neu is fusion direction-dependent.     

N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine

DNA vaccines consisted of tumor-associated antigen (TAA) are well suited for immunotherapy against tumor. The construct can contain TAA fused to an appropriate molecule (biologic adjuvant) to improve the efficacy of anti-tumor immune response. Heat shock protein 70 (HSP70) has been shown to be an excellent candidate, capable of cross-priming TAA by antigen presenting cells leading to a robust T-cell response. However, the relationship between strong T-cell responses and tumor rejection is not always mutually exclusive, for which TAA loss or activation of suppressive mechanisms may occur. HSP70 fused to downstream of Her2/neu as DNA vaccine has been shown to be efficient against Her2-expressing tumors. In this study, we examined if N-terminally fusion of Her2/neu to HSP70 could also improve efficiency of Her2/neu DNA vaccine. Therefore, mice with an established Her2/neu expressing tumor were immunized with DNA vaccine consisting of extracellular and trans-membrane domain (EC+TM) of rat Her2/neu alone or N-terminally fused to HSP70 and immune response was evaluated. Administration of rat Her2/neu led to partial control of tumor progression. Surprisingly, fusion of HSP70 to N-terminal of rat Her2/neu led to tumor progression. Our result proposes that fusion direction of biologic adjuvant is an important consideration when Her2/neu is used.     

Solamargine induces apoptosis and enhances susceptibility to trastuzumab and epirubicin in breast cancer cells with low or high expression levels of HER2/neu

Trastuzumab is used for breast cancer patients with high expression levels of HER2 (human epidermal growth factor receptor 2)/neu; however, it has no effect on cancers with low levels of HER2/neu. SM (solamargine), a major steroidal alkaloid glycoside purified from Solanum incanum, triggered apoptosis of breast cancer cells (MCF-7 and SK-BR-3 cells) and non-cancerous breast epithelial cells (HBL-100 cells) within 3 h. To extend the application of trastuzumab in breast cancer patients, the regulation of HER2/neu expression by SM was investigated. SM significantly up-regulates HER2/neu expression in breast cancer cells with low and high expression levels of HER2/neu, and synergistically enhanced the effect of trastuzumab in inhibiting cell proliferation. Additionally, HER2/neu and TOP2A [TopoII (topoisomerase II) alpha] genes share the same amplicon on an identical chromosome. Notably, SM co-regulates HER2/neu and TopoIIalpha expression markedly, and enhances TopoII inhibitor-EPI (epirubicin)-induced cytotoxicity to breast cancer cells.