Toward stable N4-modified neurotensins for NTS1-receptor-targeted tumor imaging with 99mTc

A series of Gly-neurotensin(8-13) analogues modified at the N-terminus by acyclic tetraamines (Demotensin 1-4) were obtained by solid-phase peptide synthesis techniques. Strategic replacement of amino acids and/or reduction of sensitive peptide bonds were performed to enhance conjugate resistance against proteolytic enzymes. During 99mTc-labeling, single species radiopeptides, [99mTc]Demotensin 1-4, were easily obtained in high yields and typical specific activities of 1 Ci/micromol. Peptide conjugates displayed a high affinity binding to the human neurotensin subtype 1 receptor (NTS1-R) expressed in colon adenocarcinoma HT-29 or WiDr cells and/or in human tumor sections. [99mTc]Demotensin 1-4 internalized very rapidly in HT-29 or WiDr cells by a NTS1-R-mediated process. [99mTc]Demotensin 3 and 4, which remained stable during 1 h incubation in murine plasma, were selectively studied in nude mice bearing human HT-29 and WiDr xenografts. After injection, [99mTc]Demotensin 3 and 4 effectively and specifically localized in the experimental tumors and were rapidly excreted via the kidneys into the urine, exhibiting overall biodistribution patterns favorable for NTS1-R-targeted tumor imaging in man.

The effect of rhBMP-2 around endosseous implants with and without membranes in the canine model

BACKGROUND: Bone morphogenetic protein (BMP) is a potent differentiating agent for cells of the osteoblastic lineage. It has been used in the oral cavity under a variety of indications and with different carriers. However, the optimal carrier for each indication is not known. This study examined a synthetic bioabsorbable carrier for BMP used in osseous defects around dental implants in the canine mandible. METHODS: Twelve canines had their mandibular four premolars and first molar teeth extracted bilaterally. After 5 months, four implants were placed with standardized circumferential defects around the coronal 4 mm of each implant. One-half of the defects received a polylactide/glycolide (PLGA) polymer carrier with or without recombinant human BMP-2 (rhBMP-2), and the other half received a collagen carrier with or without rhBMP-2. Additionally, one-half of the implants were covered with a non-resorbable (expanded polytetrafluoroethylene [ePTFE]) membrane to exclude soft tissues. Animals were sacrificed either 4 or 12 weeks later. Histomorphometric analysis included the percentage of new bone contact with the implant, the area of new bone, and the percentage of defect fill. This article describes results with the PLGA carrier. RESULTS: All implants demonstrated clinical and radiographic success with the amount of new bone formed dependent on the time and presence/absence of rhBMP-2 and presence/absence of a membrane. The percentage of bone-to-implant contact was greater with rhBMP-2, and after 12 weeks of healing, there was approximately one-third of the implant contacting bone in the defect site. After 4 weeks, the presence of a membrane appeared to slow new bone area formation. The percentage of fill in membrane-treated sites with rhBMP-2 rose from 24% fill to 42% after 4 and 12 weeks, respectively. Without rhBMP-2, the percentage of fill was 14% rising to 36% fill, respectively. CONCLUSIONS: After 4 weeks, the rhBMP-2-treated sites had a significantly higher percentage of contact, more new bone area, and higher percentage of defect fill than the sites without rhBMP-2. After 12 weeks, there was no significant difference in sites with or without rhBMP-2 regarding percentage of contact, new bone area, or percentage of defect fill. In regard to these three outcomes, comparing the results with this carrier to the results reported earlier with a collagen carrier in this study, only the area of new bone was significantly different with the collagen carrier resulting in greater bone than the PLGA carrier. Thus, the PLGA carrier for rhBMP-2 significantly stimulated bone formation around dental implants in this model after 1 month but not after 3 months of healing. The use of this growth factor and carrier combination appears to stimulate early bone healing events around the implants but not quite to the same degree as a collagen carrier.

Snake venom C-type lectins interacting with platelet receptors. Structure-function relationships and effects on haemostasis

Snake venoms contain components that affect the prey either by neurotoxic or haemorrhagic effects. The latter category affect haemostasis either by inhibiting or activating platelets or coagulation factors. They fall into several types based upon structure and mode of action. A major class is the snake C-type lectins or C-type lectin-like family which shows a typical folding like that in classic C-type lectins such as the selectins and mannose-binding proteins. Those in snake venoms are mostly based on a heterodimeric structure with two subunits alpha and beta, which are often oligomerized to form larger molecules. Simple heterodimeric members of this family have been shown to inhibit platelet functions by binding to GPIb but others activate platelets via the same receptor. Some that act via GPIb do so by inducing von Willebrand factor to bind to it. Another series of snake C-type lectins activate platelets by binding to GPVI while yet another series uses the integrin alpha(2)beta(1) to affect platelet function. The structure of more and more of these C-type lectins have now been, and are being, determined, often together with their ligands, casting light on binding sites and mechanisms. In addition, it is relatively easy to model the structure of the C-type lectins if the primary structure is known. These studies have shown that these proteins are quite a complex group, often with more than one platelet receptor as ligand and although superficially some appear to act as inhibitors, in fact most function by inducing thrombocytopenia by various routes. The relationship between structure and function in this group of venom proteins will be discussed.

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS: The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS: For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS: This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE: Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.

Treatment of premenopausal women with early breast cancer: old challenges and new opportunities

Breast cancer occurring in women before the age of menopause continues to be a major medical and psychological challenge. Endocrine therapy has emerged as the mainstay of adjuvant treatment for women with estrogen receptor-positive tumours. Although the suppression of ovarian function (by oophorectomy, irradiation of the ovaries or gonadotropin releasing factor analogues) is effective as adjuvant therapy if used alone, its value has not been proven after chemotherapy. This is presumably because of the frequent occurrence of chemotherapy-induced amenorrhoea. Tamoxifen reduces the risk of recurrence by approximately 40%, irrespective of age and the ovarian production of estrogens. The worth of ovarian function suppression in combination with tamoxifen is unproven and is being investigated in an intergroup randomised clinical trial (SOFT [Suppression of Ovarian Function Trial]). Aromatase inhibitors are more effective than tamoxifen in postmenopausal women but are only being investigated in younger patients. The use of chemotherapies is identical in younger and older patients; however, at present the efficacy of chemotherapy in addition to ovarian function suppression plus tamoxifen is unknown in premenopausal patients with endocrine responsive disease. 'Targeted' therapies such as monoclonal antibodies to human epidermal growth factor receptor (HER)-2, HER1 and vascular endothelial growth factor, 'small molecule' inhibitors of tyrosine kinases and breast cancer vaccines are rapidly emerging. Their use depends on the function of the targeted pathways and is presently limited to clinical trials. Premenopausal patients are best treated in the framework of a clinical trial.

Analysis and mapping of CACNB4, CHRNA1, KCNJ3, SCN2A and SPG4, physiological candidate genes for porcine congenital progressive ataxia and spastic paresis

The cause of porcine congenital progressive ataxia and spastic paresis (CPA) is unknown. This severe neuropathy manifests shortly after birth and is lethal. The disease is inherited as a single autosomal recessive allele, designated cpa. In a previous study, we demonstrated close linkage of cpa to microsatellite SW902 on porcine chromosome 3 (SSC3), which corresponds syntenically to human chromosome 2. This latter chromosome contains ion channel genes (Ca(2+), K(+) and Na(+)), a cholinergic receptor gene and the spastin (SPG4) gene, which cause human epilepsy and ataxia when mutated. We mapped porcine CACNB4, KCNJ3, SCN2A and CHRNA1 to SSC15 and SPG4 to SSC3 with the INRA-Minnesota porcine radiation hybrid panel (IMpRH) and we sequenced the entire open reading frames of CACNB4 and SPG4 without finding any differences between healthy and affected piglets. An anti-epileptic drug treatment with ethosuximide did not change the severity of the disease, and pigs with CPA did not exhibit the corticospinal tract axonal degeneration found in humans suffering from hereditary spastic paraplegia, which is associated with mutations in SPG4. For all these reasons, the hypothesis that CACNB4, CHRNA1, KCNJ3, SCN2A or SPG4 are identical with the CPA gene was rejected.

Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves

Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs

Does the beta-blocker nebivolol increase coronary flow reserve?

INTRODUCTION: Nebivolol, a highly selective beta1-adrenergic receptor-blocker, increases basal and stimulated endothelial nitric oxide (NO)-release. It is unknown, whether coronary perfusion is improved by the increase in NO availability. Therefore, we sought to evaluate the effect of nebivolol on coronary flow reserve (CFR) and collateral flow. METHODS: Doppler-flow wire derived coronary flow velocity measurements were obtained in ten controls and eight patients with coronary artery disease (CAD) at rest and after intracoronary nebivolol. CFR was defined as maximal flow during adenosine-induced hyperemia divided by resting flow. In the CAD group, collateral flow was determined after dilatation of a flow-limiting coronary stenosis. Collateral flow index (CFI) was defined as the ratio of flow velocity during balloon inflation divided by resting flow. RESULTS: CFR at rest was 3.0+/-0.6 in controls and 2.1+/-0.4 in CAD patients. After intracoronary doses of 0.1, 0.25, and 0.5 mg nebivolol, CFR increased to 3.4+/-0.7, 3.9+/-0.9, and 4.0+/-0.1 (p<0.01) in controls, and to 2.3+/-0.7, 2.6+/-0.9, and 2.6+/-0.5 (p<0.05) in CAD patients. CFI decreased significantly with intracoronary nebivolol and correlated to changes in heart rate (r=0.75, p<0.001) and rate-pressure product (r=0.59, p=0.001). DISCUSSION: Intracoronary nebivolol is associated with a significant increase in CFR due to reduction in resting flow (controls), or due to an increase in maximal coronary flow (CAD patients). CFI decreased with nebivolol parallel to the reduction in myocardial oxygen consumption.

Chemokine receptor 4 (CXCR4) is part of the lipopolysaccharide "sensing apparatus"

Recognition of bacterial lipopolysaccharide (LPS) by the innate immune system involves at least three receptor molecules: CD14, TLR4 and MD-2. Additional receptor components such as heat shock proteins, chemokine receptor 4 (CXCR4), or CD55 have been suggested to be part of this activation cluster; possibly acting as additional LPS transfer molecules. Our group has previously identified CXCR4 as a component of the "LPS-sensing apparatus". In this study we aimed to elucidate the role that CXCR4 plays in innate immune responses to LPS. Here we demonstrate that CXCR4 transfection results in responsiveness to LPS. Fluorescence correlation spectroscopy experiments further showed that LPS directly interacts with CXCR4. Our data suggest that CXCR4 is not only involved in LPS binding but is also responsible for triggering signalling, especially mitogen-activated protein kinases in response to LPS. Finally, co-clustering of CXCR4 with other LPS receptors seems to be crucial for LPS signalling, thus suggesting that CXCR4 is a functional part of the multimeric LPS "sensing apparatus".

Medroxyprogesterone abrogates the inhibitory effects of estradiol on vascular smooth muscle cells by preventing estradiol metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.

cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

The role of interleukin-6 in the angiogenesis of ovarian carcinoma

The purpose of this study was to characterize the effects of IL-6 on endothelial cells and to investigate the role of IL-6 in the angiogenesis of ovarian carcinomas. We evaluated human ovarian carcinoma clinical specimens and determined that high expression of IL-6 was associated with increased tumor vascularization. Additionally, endothelial cells derived from the ovary and mesentery expressed the IL-6 receptor (IL-6R), and their stimulation with the exogenous ligand activated downstream signaling molecules and enhanced cell migration. Dual immunohistochemical staining for CD-31 and IL-6R revealed IL-6R expression on human endothelial cells within normal ovary and ovarian carcinomas. To further investigate the possible proangiogenic function of IL-6, Gelfoam sponges containing IL-6 or bFGF were implanted into the subcutis of BALB/c mice. IL-6 containing sponges were vascularized to the same extent as bFGF containing sponges. ^ Chronic stress can adversely affect disease progression. Stimulation of ovarian carcinoma cell lines with concentrations of catecholamines achieved in individuals experiencing chronic stress resulted in a substantial increase in IL-6 production. It was determined that stress mediators regulate IL-6 expression through the β-adrenergic receptor and Src. These data illustrate one mechanism by which chronic stress may influence tumor progression. ^ To investigate whether IL-6 contributes to the angiogenesis of ovarian carcinomas, we isolated low IL-6 expressing clones from the SKOV3.ip1 cell line and transfected them with a plasmid encoding the IL-6 gene. We observed no difference in tumor weight between high and low IL-6 expressing cells. However, while low IL-6 expressing tumors were highly vascularized, high IL-6 expressing tumors appeared hypervascularized. Immunohistochemical analysis revealed that all tumors exhibited robust expression of additional proangiogenic molecules. ^ Collectively, these studies indicate that IL-6 secreted by ovarian cancer cells is a highly proangiogenic cytokine. However, IL-6 is but one of several proangiogenic molecules produced by ovarian cancer, and its inhibition may not be sufficient to inhibit angiogenesis of ovarian carcinoma. The findings presented in this dissertation provide insight into the function of IL-6 as a regulator of angiogenesis. Understanding of the role of proangiogenic molecules such as IL-6 in ovarian carcinoma may have important implications for therapy directed at the vascular component of this disease. ^

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER Midan Ai, Ph.D. Supervisory Professor: Zhen Fan, M.D. Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism needs to be determined. In my studies, I found that high expression of HER2 is correlated with high expression of Brk in breast cancer cell lines. Silencing HER2 expression via RNA interference in HER2 over-expressed breast cancer cells resulted in Brk protein decrease and overexpression of HER2 in HER2 low-expressed breast cancer cells up-regulated Brk expression. The mechanism study indicated that overexpression of HER2 increased Brk protein stability. Brk was degraded through a Ca2+-dependent protease pathway involving calpain and HER2 stimulated Brk expression via inhibiting calpain activity. Calpastatin is a calpain endogenous inhibitor and the calpain-calpastatin system has been implicated in a number of cell physiological functions. HER2 restrained calpain activation via up-regulating calpastatin expression and HER2 downstream signaling, MAPK pathway, was involved in the regulation. Furthermore, silencing of Brk expression by RNA interference in HER2-overexpressing breast cancer cells decreased HER2-mediated cell proliferation, survival, invasion/metastasis potential and increased cell sensitivity to HER2 kinase inhibitor, lapatinib, treatment, indicating that Brk plays important roles in regulating and mediating the oncogenic functions of HER2. The Stat3 pathway played important roles in Brk mediated cell survival and invasion/metastasis in the context of HER2-overexpressing breast cancer cells. However, transgenic mice with inducible expression of constitutively active Brk (CA) in the mammary epithelium failed to develop malignant change in the mammary glands after Brk induction for 15 months which indicated that expression of Brk protein alone was not sufficiently to induce spontaneous breast tumor. Bitransgenic mice with co-expression of HER2/neu and inducible expression of Brk in the mammary epithelium developed multifocal mammary tumors, but there were no significant difference in the tumor occurring time, tumor size, tumor weight and tumor multiplicity between the mouse group with co-expression of Brk and HER2/neu and the mouse group with HER2/neu expression only.